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Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential.

The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.

Observations on the vegetation of northeastern Mato Grosso II. Forests and soils of the Rio Suiá--Missu area.

The vegetation of the well drained soils along the Suiá--Missu road in the Serra do Roncador region of NE Mato Grosso is Evergreen Seasonal forest of Amazonian type. The area lies close to the meeting place of the Amazonian forest (the hylaea) and the cerrado (savanna) formation of Central Brazil. The structure of the forest is simple: the canopy is at about 18--23 m, and is exceeded by a few scattered emergents; no recognizable strata can be distinguished among the understorey trees and the shrub and herb layers are sparse. Table 1 lists the most important species and gives information on stratification and general distribution. Most of the species appear to have a hylaean centre of distribution but extend into other vegetation types. The forest differs from related communities which lie closer to the cerrado/forest boundary in its greater height and luxuriance, the presence of additional tall tree species, and the great reduction in abundance of a cerrado floristic element. A survey on the Xavantina--São Felix road allowed us to extend previous observations on the distance to which the cerrado tree Pterodon pubescens extends into the forest. The results obtained indicate a considerable extension of forest into cerrado during the life of an individual tree. A characteristic low forest occurs in the flood plain of the Rio Suiá--Missu while Swampy Gallery forests occur on permanently waterlogged soils around the headwaters of streams. The well drained soils of the Suiá--Missu forest are very uniform, deep latosols (oxisols) of very dystrophic nature with pH (in water) between 4.0 and 5.0 (see table 2, p. 203).

Urodynamic studies in prune belly syndrome. A case report.

Urodynamic studies were carried out in a 14-year-old boy with Prune Belly Syndrome and terminal renal failure prior and after successful renal transplantation. Increased bladder capacity, nonprovocative detrusor instability and a high compliance were the most characteristic findings during the filling phase of the bladder. During the voiding phase an increased detrusor pressure was demonstrated. Outflow resistance and maximum urinary flow rate were within normal range before and after transplantation. In contrast to the findings before renal transplantation, however, micturition was imbalanced after transplantation (residual urine 100 ml). Urodynamics revealed that the bulging of the posterior urethra, observed in the early voiding phase, was due to a congenital insufficiency of the posterior urethral musculature (megalourethra) and not caused by mechanical obstruction leading to urethral dilatation. It is suggested that detrusor-bladder-neck-dyssynergia is the primary cause of the imbalanced micturition and its consequences (bladder distention, reflux, urinary tract infection, hydronephrosis, pyelonephritis) in patients with Prune Belly Syndrome. The findings of a normal, respectively increased detrusor activity are in contrast to the observations of some authors, describing attenutation and absence of detrusor muscle fibres. The indications and effects of transurethral resection and internal urethrotomy, proposed by some authors, are discussed.

Nervous control of pancreatic exocrine secretion in pigs.

The pancreatic secretion of fluid, bicarbonate and protein in response to electrical stimulation of the vagus and splanchnic nerves, to exogenous and endogenous secretin and to various pharmacological agents was studied in anesthetized young pigs (21 kg). Vagal stimulation increased flow, bicarbonate output and protein output in a frequency dependent manner; the half maximal effective frequency was 2--4 Hz and the maximal effective frequency 12 Hz. The secretory response to vagal stimulation was potentiated by physiological elevations of the arterial concentration of secretin brought about by injection of secretin or by acidification of the duodenal bulb. Simultaneous stimulation of the splanchnic nerves strongly inhibited the response to vagal stimulation; splanchnic nerve stimulation alone had no demonstrable effect. The flow and bicarbonate response to vagal stimulation was unaffected by atropine, but abolished by hexamethonium. Protein output was strongly inhibited by both agents. The response to intraarterial infusion of acetylcholine resembled that elicited by vagal stimulation but it was smaller and it was completely abolished by atropine and unaffected by hexamethonium. Alpha- and beta-adrenergic blockade stimulated rather than inhibited the secretory response to vagal stimulation. The portal vein plasma concentration of secretin was not affected by vagal stimulation. The results indicate that the protein response, and the flow and bicarbonate response to vagal stimulation are not brought about by the same mechanism. An increased release of secretin is not involved. Peptidergic (VIP-containing) nerves may contribute.

Genetic alterations of Streptococcus mutans' virulence.

The use of mutants defective in caries-associated traits has enabled the genetic dissociation of agglutination from adhesion, the demonstration of serotype-specific contributions of IPS to virulence, the importance of glucanohydrolase to virulence to a greater degree than to plaque formation, and the apparent lack of importance of agglutination to virulence. We have also been able to demonstrate the ability of plaque formation-defective mutants and other variants both to infect and to emerge, yet not to cause disease. Additional mutants, currently under study in our laboratory include fructanase, invertase, and sucrose permease-defectives. Ultimately, the identification of key, probably surface-associated virulence factors will offer more potent and specific antigens for directed immune responses by the host.

[Diagnosis of breast cancer by contact thermography (author's transl)].

Epidemiological studies performed in cooperation with the Austrian National Institute for Statistic show an increase of breast cancer in the age-group of women older than 50 years. Especially in this age-group screening for breast cancer is very important and successful. Mammography in this age-group is the most useful and accurate method. In the group of women aged less than 50 years and particularly less than 45 years the use of mammography for screening is limited because of its risks for the induction of breast cancer in young women. Therefore the combination of clinical examination and contact-thermography is a very promising alternative for the detection of breast cancer. In 133 cases of histologically proved breast cancer the high accuracy of this combination of clinical examination and contact-thermography in comparison with mammography is shown. Based on this good results a recommendation for a detection-scheme for breast cancer with a reduction of radiation exposure in young women by the use of contact-thermography is presented and discussed.

First experience with continuous pH measurements on fetus during delivery.

First experience with continuous pH measurement is reported. It was possible to apply well calibrated electrodes to the fetal scalp in 22 cases. Of these 22 cases eight must be regarded as failures because of unstable values. Of the remaining 14 measurements, seven were of good (or acceptable) quality and seven of poor quality. The size of the electrodes and the excessive depth of penetration are probable reasons for these failures. An important condition for obtaining reliable pH values is a perpendicular position of the electrode in the fetal scalp. An indicator for correct position of the electrode appears to be the irregularity in the recording of the pH curve. Experience so far indicates that the pH electrode in its present form is not suitable for routine use.

Improvements in the results with the continuous pH electrode due to technical progress: a comparison between two series of cases.

Our experience is based on 139 attempts of tissular pH electrode applications in the fetal scalp during labor and includes 119 records comprising 86 tracings of good quality and 33 tracings of bad quality. Technical progress in the electrode and application material allowed an increase in the good quality tracings rate (from 54.5--66%) and a decrease of the complications, particularly the numbers of broken electrodes (from 18 to 2.4%).

A comparative approach to protein- and ligand-dependence of the Root effect for fish haemoglobins.

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller.

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8--4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.

Isolation and characterization of Ca2+-dependent modulator protein from the marine invertebrate Renilla reniformis.

An acidic, low molecular weight (18 400--19 100) protein capable of activating porcine brain phosphodiesterase in the presence of calcium has been purified 2700-fold from the anthozoan coelenterate, Renilla reniformis. The protein has physical, spectral, and chemical properties similar to those of modulator proteins isolated from mammalian species. Amino acid composition studies reveal no significant differences between the Renilla and mammalian modulator proteins. For example, we observed 1 mol of epsilon-N-trimethyllysine per mol of protein, no tryptophan or cysteine, and high levels of glutamic and aspartic acid residues. The protein from Renilla complexes with troponin I and T subunits in the presence of calcium and quantitatively replaces porcine brain modulator in the calcium-dependent activation of porcine brain phosphodiesterase. The protein has a high affinity for calcium as judged by the low levels of free calcium required for modulator-dependent activation of phosphodiesterase. The similarities in physical and chemical properties, high affinity for calcium, and identical calcium-dependent activities of this protein from Renilla (as compared with modulator protein purified from mammalian systems) suggest that a high degree of structural conservation has been retained in modulator proteins isolated from these diverse evolutionary forms.

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.

Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats.

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6.) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, Pi and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10--20% of its maximum capacity.

Comparative study of automatic blood-gas analysers and their use in analysing arterial and capillary samples.

Three automatic blood-gas analysers were compared for ease of use; calibration; reproducibility and accuracy of results; maintenance; fault-finding; and use of expert technician time. Results obtained from arterial and capillary blood were compared with duplicate values obtained with a semi-automatic analyser controlled and calibrated with tonometered blood. No analyser was fully automatic, and all three needed maintenance by expert technicians. Difficulties were encountered when inexperienced operators used the machines. One automatic blood-gas analyser gave aberrant values for oxygen pressure (PO2) due to electrode dysfunction that was not indicated by the fault-finding system. A second analyser gave significantly lower values for blood pH than the standard machine. A comparison of pH, carbon dioxide pressure (PCO2), and PO2 measured in 40 simultaneous paired samples of arterial and arterialised capillary blood showed no significant difference for pH or PCO2, but the PO2 values were significantly lower in the capillary samples over the range studied. We conclude that all machines perform satisfactorily in terms of blood-gas analysis, but none may be regarded as fully automatic. Some degree of technical supervision is essential, as is proper training for all potential users.

A study of carbachol-atropine interaction on intestinal smooth muscle vesicles, using a fluorescent probe.

Plasma membrane vesicles were prepared from guinea pig ileum longitudinal muscle. The vesicles were characterized by electron microscopy and analysis of lipid and protein content. They were shown to be free of gross contamination from actomyosin, sarcoplasmic reticulum, and mitochondria. 8-Anilino-1-naphthalene sulphonic acid (ANS) binding characteristics were similar to those found in other membranes. Both carbachol and atropine increased the fluorescence of ANS bound to this membrane, the maximum increase for atropine being greater than that for carbachol. Since neither drug effected the apparent affinity constant for the ANS-membrane interaction. It may be assumed that the increased fluorescence was due to an increase in the number of ANS binding sites. The carbachol-dependent increase in ANS fluorescence was blocked noncompetitively by atropine but not by tubocurarine or diphenhydramine. These latter two antagonists also increased ANS fluorescence but at much higher concentrations than either carbachol or atropine. Neither atropine nor carbachol increased ANS fluorescence on either erythrocyte ghosts or liposomes (prepared from a lipid extract of the muscle membrane).

Neuropharmacological regulation of episodic growth hormone and prolactin secretion in the rat.

The effects on GH and PRL secretion of several pharmacological agents known to modify central neurotransmitter action were determined in unanesthetized male rats. Phenoxybenzamine, an alpha-adrenergic blocker (5 mg/kg iv), abolished episodic GH secretion and caused elevation of serum PRL levels. Propranolol, a beta-adrenergic blocker (5 mg/kg iv), had no effect on GH secretion and caused a small but significant depression in PRL levels. Parachlorophenylalanine methyl ester, an inhibitor of tryptophan hydroxylase (300-350 mg/kg ip), resulted in significant inhibition of GH pulsatile secretion and suppressed PRL levels. Methysergide hydrogen maleinate (25 mg/kg iv), a serotonin receptor antagonist, also inhibited GH secretion, but produced a transient stimulation in PRL levels. Atropine sulfate (2 mg/kg iv) caused significant suppression in GH secretion, but had no effect on PRL. Picrotoxin, a gamma-aminobutyric acid antagonist, in a subconvulsive dose of 1-3 mg/kg iv, also depressed GH episodic secretion but did not affect PRL levels. These results indicate that several neurotransmitters, i.e., norepinephrine, serotonin, acetylcholine, and gamma-aminobutyric acid, found in high concentration in the hypothalamus, influence GH and PRL secretion.

On the role of calcium in adrenocorticotropin-induced changes in mitochondrial pregnenolone synthesis.

The importance of calcium in the ACTH-induced increase in adrenal mitochondrial pregnenolone synthesis was evaluated. In mitochondria prepared in the absence of EDTA and albumin, calcium enhanced the binding of cholesterol to cytochrome P-450 and subsequent pregnenolone synthesis. Although these effects of calcium were slightly greater in control than in ACTH-treated mitochondria, a sizeable effect of ACTH remained even at high calcium levels (500 micron). In mitochondria prepared from adrenals homogenized in fluid containing EDTA and albumin, ACTH-induced effects on pregnenolone synthesis were relatively poor unless calcium was added to the incubation mixture. High concentrations of added calcium (500 micron or greater) obviated the need for the labile protein required for ACTH-induced effects in intact mitochondria, presumably by disrupting mitochondria and allowing an "unrestrained" interaction of cholesterol with cytochrome P-450. Thus, cholesterol-rich mitochondria from ACTH plus cycloheximide-treated rats produced large amounts of pregnenolone when high (probably unphysiological) calcium concentrations were present. The present findings suggest that calcium is required at the mitochondrial level for ACTH-induced effects on pregnenolone synthesis, and the reported ACTH-induced increase in intraadrenal calcium may thus amplify the effects of ACTH on steroidogenesis. However, it seems unlikely that calcium is the agent primarily responsible for mediating the ACTH-induced steroidogenic effect at the mitochondrial level.

Alterations in blood levels of carbohydrate and lipid metabolites and of cyclic AMP mediated by beta1- and beta2-adrenoceptors in beagle dogs: effects of procaterol, a new selective beta2-adrenoceptor agonist.

The intravenous injection of isoprenaline (10 nmole/kg) into conscious beagle dogs caused significant increases in the blood level of lactate, glucose, FFA, insulin and cyclic AMP. These metabolic alterations induced by isoprenaline were blocked completely by pretreatment of the dog with propranolol (1 mg/kg). Butoxamine (10 mg/kg) antagonized isoprenaline-induced increases in glucose, lactate and insulin, but not the increases in FFA. Practolol (10 mg/kg) diminished the increase in blood FFA very strongly. Salbutamol, which is known to be an agonist of the beta 2-subtype in its bronchomotor and cardiovascular actions, produced marked increases in the blood concentrations of lactate, glucose and insulin but were without effect on the FFA level. Thus metabolic responses of conscious beagle dogs to beta-adrenoceptor agonists appeared to depend differentially on two types of beta-adrenoceptors: beta1-adrenoceptors are largely involved in lipolysis while beta2-adrenoceptors are involved in the regulation of blood glucose metabolism and insulin secretion. A new beta-adrenoceptor agonist, 5-(1-hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostyril hydrochloride hemihydrate (Procaterol), was classified as a beta 2-agonist, because it markedly increased plasma concentrations of glucose, lactate and insulin but increased the plasma level of FFA to a lesser degree. The order of potency of beta2-agonists was procaterol greater than salbutamol greater than trimetoquinol. Metabolic responses of beagle dogs would be useful for appreciating the selectivity and potency of beta-adrenoceptor agonists and antagonists.

[Report on the round-table discussion of the subject "idea and experience" in connection with the 1977 annual meeting "process kinetics" of the German Academy of Natural Scientists Leopoldina (Halle/S., October 16, 1977)].

A report is given about a round-table-discussion upon the role and meaning of "idea and experience" in the creative scientific process. Notable representatives of mathematics, theoretical physics and geophysics, chemistry, theoretical and general experimental biology, and of medicine contribute in the discussion guided by C. F. V. WEIZSACKER to this theme from point of view of their disciplines. The components of meaning of "idea and experience" in their connection one to another may be paraphrased by such pairs of terms as "theory and practice", "theoretical or empirical", "law and appearance of a single phenomenon", "unity and diversity", abstract and concrete". It was demonstrated that in each of the mentioned scientific disciplines there is a natural, and that, starting from mathematics and going to biology and medicine, the weight in that relation will shift more and more to "experience". Many of the known methodological problems and difficulties will arise in the mentioned scientific branches if one stresses immoderately only one component of "idea and experience" by leaving the natural, discipline-related range of variation of the relation "idea and experience".

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.

Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan protein complex.

Treatment of cells grown to exponential phase with 4% sodium dodecyl sulfate for 3 h at 100 degrees C resulted in solubilization of all cellular components except for peptidoglycan. In most strains, cells cultured in liquid gonococcal broth at pH 7.2 yielded a peptidoglycan composed primarily of N-acetylmuramic acid N-acetylglucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1:1:2:1:1. The peptidoglycan in these cells accounted for 1 to 2% (dry weight) of the cells. However, in cells cultured at pH 6.0, the dry weight of peptidoglycan increased to 4 to 13%. Preliminary investigations indicated that the apparent increase in weight is strain dependent and is due in part to associated protein(s). Neisseria gonorrhoeae strain CS7 had elevated amounts of protein associated with the peptidoglycan regardless of growth pH. The peptidoglycan-protein complex could not be dissociated by additional extraction with sodium dodecyl sulfate, 10 M LiCl2, or ethylenediaminetetraacetate or by 7.5% polyacrylamide gel electrophoresis. The complex could be degraded by lysozyme, trypsin, chymotrypsin, Pronase B, and Chalaropsis sp. muramidase.

The "unexplained" poor postcoital test.

In 30 or 32 infertile couples with an unexplained negative or bad in vivo and in vitro sperm penetration test, we obtained a strongly positive Sperm Cervical Mucus Contact test (SCMC-test) and demonstrated the presence of antisperm antibodies in the male or female partner. In these 30 couples 25 of the male partners had a sperm-agglutination titre of at least 32 in the serum and at least 4 in the seminal plasma. In the five remaining couples the female partner showed a minimum sperm-agglutination titre of 16 in the serum and a cervical mucus titre of at least 128. In 48 couples with a fair or good sperm penetration in cervical mucus, in vivo and in vitro, we never found a strongly positive SCMC-test. In 43 of these couples the SCMC-test was negative. Only one man in the latter group had sperm-agglutinating activity in the semen. In a group 32 couples, with a negative SCMC-test, there was no or only weak sperm-agglutinating activity in the cervical mucus, although 2 women had moderate sperm-agglutinating activity in the blood serum. Based on these data we conclude that the so called "unexplained" poor postcoital test is almost always due to the presence of antisperm antibodies in the semen or in the cervical mucus. We consider the SCMC-test not only to be a simple and reliable technique for detecting the presence of these antisperm antibodies, but also a method of demonstrating the mechanism by which antisperm antibodies decrease the chance of conception.

Haloacetyl groups as reversible protection of the amino function: cleavage with 2-aminothiophenol.

Haloacetylamino acids and haloacetyl peptides react rapidly with 2-aminothiophenol in weakly alkaline media to yield 2-aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N-haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloroacetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates. Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2-haloacids to modern polypeptide chemistry are outlined.

Peptidoglycan synthesis in cocci and rods of a pH-dependent, morphologically conditional mutant of Klebsiella pneumoniae.

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.

Possible involvement of bacterial autolytic enzymes in flagellar morphogenesis.

Autolytic enzymes were found to be required for flagellar morphogenesis in Bacillus subtilis 168 and Bacillus licheniformis 6346. Two previously characterized, poorly lytic, chain-forming mutants of B. subtilis 168, strains FJ3 (temperature conditional) and FJ6, each 90 to 95% deficient in the production of N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase, were observed to be nonmotile at 35 degrees C in a variety of liquid and semisolid meida. In contrast, cells of the isogenic wild-type strain were motile and fully separated. Electron microscopy revealed the complete absence of flagella on the mutant cells. Similar observations were made with another poorly lytic strain of B. subtilis 168 (Nil5) and with two poorly lytic, phosphoglucomutase-deficient mutants of B. licheniformis 6346 (MH-3, MH-5). In minimal media lacking galactose (restrictive conditions), the B. licheniformis mutants failed to form flagella, or had serious abnormalities in flagellar morphogenesis and motility. Under permissive conditions, mutants FJ3 (grown at 17 degrees C) and MH-5 (grown with addend galactose) showed increased autolytic activities, grew in the dechained form, and regained their capacities to synthesize functional flagella. Examination of several classes of spontaneous revertants derived from the various mutant strains further demonstrated a close relationship between autolysin acttivity and flagellation in the two Bacillus spp.

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.

Splenectomy and sudden death.

Four cases of fatal fulminant Streptococcus pneumoniae septicemia in asplenic individuals have been presented, demonstrating the relative lack of specificity of the symptoms and rapidity of the clinical course. Vigorous specific therapy was without apparent effect in two of the cases. No apparent reticuloendothelial deficiency prior to splenectomy was detected in two cases, and theoretically rather than clinically present in the others. Individual was hyposplenic secondary to splenic atrophy. The rapidity of the course and unexpected death will often bring such cases under the jurisdiction of the coroner or medical examiner, and medicolegal investigators should be alert for this syndrome.

Physicochemical properties of amphoteric beta-lactam antibiotics. II: Solubility and dissolution behavior of aminocephalosporins as a function of pH.

The solubility of aminocephalosporins in aqueous solution at 37 degrees and an ionic strength of 0.5 exhibited U-shaped curves against pH. At their isoelectric pH, cephradine monohydrate was the most soluble, followed by cephalexin monohydrate and cephaloglycin dihydrate, with intrinsic solubilities of 26.0, 17.2, and 14.8 mg/ml, respectively. The dissolution rate constants from the rotating disk were also determined as a function of the pH of the dissolution medium and interpreted reasonably by the simultaneous dissociation equilibrium reaction and the diffusion kinetics model. Energies for the solubility and dissolution were determined for these three aminocephalosporins.

An investigation of tensile strength of dental solder joints.

Tensile specimens of dental solder joints were tested to determine the effect of gap distance on the strength of solder joints. Two dental alloys were used; a Type III gold alloy and a gold-palladium (Au-Pd) alloy. The Type III alloy was joined with the recommended gold solder. The Au-Pd alloy was joined using preceramic and postceramic application soldering techniques. Gap distances of 0.13 mm, 0.5 mm, and 1 mm were used with each soldering technique. All solder specimens were machined to a uniform diameter and then tested for tensile strength. It was concluded that: 1. There was a significant increase in strength for the Type III gold as the gap distance was increased. 2. The trend toward stronger joints at narrow gap distances for Au-Pd alloy joined with presolder was not statistically significant. 3. The trend toward stronger joints at wide gap distances for the Au-Pd alloy joined with the postsolder was not statistically significant. 4. The postsolder joints were statistically stronger than the presolder joints. 5. The trends observed could be partially explained in terms of competing effects of yield strength (triaxially), wettability, and void or inclusion content at the various gap distances.

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.

Adaptive changes induced by high altitude in the development of brain monoamine enzymes.

Exposure to high altitude (HA) affects neurotransmitter levels in the adult brain and induces a number of neurologic and behavioral disturbances. The present work was undertaken to investigate the effects of chronic exposure to a moderate hypoxic environment (natural altitude of 3800 m, 12.8% O2 in inspired air) on the development from birth until adulthood of brain monoamine enzymes in rats. The activity of synthesizing (tyrosine and tryptophan hydroxylase) and catabolizing (catechol-O-methyl transferase and monoamine oxidase) enzymes were studied in discrete brain areas (cerebral cortex, cerebellum, mesodiencephalon, hypothalamus, corpus striatum, and pons medulla) and was shown to be selectively affected by HA, depending on the age of the animal and the brain region. In general, enzyme activity was less susceptible to HA during the first week after birth than at later ages, some brain areas such as the hypothalamus showing significant alterations in some enzymes throughout development, and in all enzymes at adulthood. Furthermore, in all brain areas and at all ages, tyrosine and tryptophan hydroxylase were more affected by HA than the catabolizing enzymes, and their activity was increased in some areas (e.g., cerebral cortex and cerebellum) but decreased in other areas (e.g., hypothalamus, mesodiencephalon, corpus striatum). These enzymatic changes and the corresponding alterations in precursor amino acids, particularly tryptophan, seem to be due more to the direct effect of hypoxia on oxygen-dependent enzymes, than to the stress. It appears that an hypoxic environment may provoke both early and long-term alterations in catecholamine and serotonin metabolism, thus neurotransmitter imbalances may explain some of the alterations in neurologic and endocrine development characteristic of the hypoxic animal.

Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments.

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of clathrin with contractile proteins. Muscle G-actin, F-actin, and alpha-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris-HCl (pH 7.5) were found to be elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCl resulted in the dissassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

Appearance of catecholamine-synthesizing enzymes during development of rat sympathetic nervous system: possible role of tissue environment.

We sought to determine, in rat embryo, when and at what site in their migration cells derived from the neural crest differentiate into sympathetic neuroblasts. This has been accomplished by immunocytochemical detection, within the cells, of the enzymes catalyzing catecholamine biosynthesis-tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] dopamine-beta-hydroxylase [DBH; 3,4-dihydroxyphenylethylamine,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1)]-and, as a marker of prospective adrenal medullary cells, the enzyme phenylethanolamine N-methyltransferase (PNMT; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28). TH and DBH, not detected in the neural crest, appear almost simultaneously in cells of the thoracic sympathetic ganglia in 11-day-old embryos, and in abdominal and lumbar ganglia 1-2 days later, thereby exhibiting a characteristic rostral-caudal gradient of differentiation. Cells stained for TH and DBH are seen in the gut wall from day 11 to day 14, but not thereafter. Cells stained for TH and DBH appear in the adrenal anlage at day 15. However, PNMT is not detected in the adrenal until day 17 of development, and is present only in the sympathoblasts in contact with the adrenal cortex. Treatment of pregnant rats with dexamethasone failed to accelerate the appearance of PNMT in the embryo or to initiate its expression in cells of other sympathetic organs. We conclude that neural crest cells express a noradrenergic phenotype only after leaving the neural crest and that these cells are labile with respect to their neurotransmitter and are capable of transformation in response to environmental stimuli.

Ionic basis of different synaptic potentials mediated by an identified dopamine-containing neuron in Planorbis.

A specified dopamine neuron in Planorbis corneus produces dopamine-mediated e.p.s.ps, i.p.s.ps or biphasic, depolarizing-hyperpolarizing p.s.ps in different follower neurons. The excitatory potentials were of three types. Some follower neurons exhibited slow e.p.s.ps (ca 1 s), and a long-lasting, slowly desensitizing, depolarizing response to iontophoresed dopamine. Others showed rapid (ca. 150 ms) e.p.s.ps, often of variable amplitude, and a rapid, quickly desensitizing, response to iontophoresed dopamine. The rapid e.p.s.ps were sometimes followed by the inhibitory response (biphasic potential). The e.p.s.ps were potentiated by hyperpolarization and reduced by depolarization, though they could not be inverted. The slow e.p.s.p. was shown to be associated with an increase in membrane conductance, but it has proved difficult to elucidate the ions involved. A third type of e.p.s.p. was produced by electrical transmission. The inhibitory potentials were generally reduced in amplitude by artificial hyperpolarization but could rarely be inverted. This is probably due in part to the presence of of electrotonic coupling between these follower neurons. The i.p.s.ps were associated with an increase in conductance which appeared small when measured in the cell body. However, the i.p.s.ps produced considerable shunting of electrotonic transmission between coupled followers indicating a large increase in conductance at the synapse. I.p.s.ps were unaffected by Cl-free solution but they were greatly reduced, though rarely inverted, by increasing the external K concentration. They were blocked by intracellular tetraethylammonium, or cooling. The effects on corresponding responses to iontophoresed dopamine were in each case the same as on the i.p.s.ps. It is concluded that the i.p.s.ps mediated by the dopamine neuron are produced by an increase in permeability to K+. On a few occasions i.p.s.ps mediated by the dopamine neuron were potentiated by hyperpolarization. This appeared to be caused by a sharp increase in membrane resistance with hyperpolarization of these particular neurons. However, mediation by a mechanism of conductance decrease could not be completely excluded.

Long-term prognosis and followup in schizophrenia.

The long-term course or natural history of schizophrenia is correlated with differing diagnostic criteria and commonly agreed upon prognostic variables. A review of 38 long-term followup studies of hospitalized schizophrenics reveals that unspecified or Kraepelinian-type schizophrenia has a much worse prognosis than atypical schizophrenia, schizoaffective psychosis, reactive psychosis, or other good premorbid types. Diagnoses based on longitudinal as well as cross-reactional data are more predictive of outcome than cross-sectionally based diagnoses. Drug and psychosocial treatment results must be evaluated in terms of prognostic variables, many of which are incorporated in some currently employed diagnostic criteria. There is no firm evidence that maintenance medication is indicated in some good prognosis patients. The paucity of long-range followups, the inadequacies of outcome assessments, and diagnostic disagreements limit our understanding of the effects of drug treatment, a treatment which is not without dangerous neurological side effects in many patients.

Mediatory role of calcium and guanosine 3', 5'-monophosphate in adrenocorticotropin-induced steroidogenesis by adrenal cells.

When incubated in a calcium-free medium, isolated rat fasciculata cells showed neither an increase in the concentration of guanocine 3',5'-monophosphate (cyclic GMP) nor an increase in corticosterone production in response to adrenocorticotropic hormone (ACTH). In response to submaximum and maximum steroidogenic concentrations of ACTH, corticosterone formation was directly proportional to increases in calcium concentration ranging from 0 to 2.5 mM. Higher concentration of calcium, however, inhibited maximal ACTH-induced steroidogenesis. In the absence of ACTH, calcium did not stimulate cyclic GMP accumulation and corticosterone formation. ACTH-induced corticosterone synthesis, preceded by an increase in cyclic GMP, was restored when ACTH and calcium were both present in the medium. Cyclic GMP or dibutryl cyclic GMP-induced steroidogenesis was substantially reduced in the absence of calcium, but in contrast to the ACTH effect a significant amount of corticosterone formation occurred without calcium. It is proposed that at the physiological concentrations of the hormone, calcium regulates the transduction of information between hormone receptors and guanylate cyclase.

[Evaluation and comparison of acid-base findings obtained using the partial and Astrup's technic in cattle].

The aim of the study was to compare the acid-base (AB-) findings in cattle obtained parallelly by the equilibration method after Astrup and the partial method (titration determination of total carbon dioxide in blood plasma) and at the same time to determine pH, net acid-base exudate in urine and the specific weight of urine. Altogether we made 65 parallel examinations in three groups of cows according to phases of reproduction. In 70.8% of cases correlation between the two results was very good or good, however under the condition that in many cases it was necessary to know also blood pH for the employment of the partial method. Without the knowledge of the blood pH good correlation would be obtained only in 33.8% of all cases. The partial method does not provide satisfactory results mainly in diagnosing respiratory disturbances and in determining compensatory degree of acid-base disturbances. We analysed two alternative values for the top, reference limit of vol. % CO2 in plasma: 60.0 or 55.0. The results imply that from the aspect of correct partial method interpretation it is more accurate to use the value 60.0. A high interpretation correlation between pH and net acid-base urine exudate was also confirmed and that independent urine pH values may be used in a complex acid-base examination to obtain acid-base findings. The representation of the basic types of acid-base disturbances indicates that metabolical acidoses are most frequent in highly pregnant cows (36.9%) mostly without proved compensation. The largest proportion of normal acid-base findings (43.4%) was determined in cows after calving in which also the compensatory mechanisms were most often activated. In connection with these findings the possibilities of impaired health of calves are discussed.

5 alpha-Reductase activity in human placenta.

The concentration of 5 alpha-pregnane-3,20-dione in peripheral blood of pregnant women is higher than that found in blood obtained from nonpregnant women throughout the luteal phase of the ovulatory cycle. The in vitro synthesis of 5 alpha-reduced pregnanes from [3H]progesterone by placental tissue was investigated using tissue minces and homogenates in the presence of added nicotinamide-adenine dinucleotide phosphate. The metabolites, [3H]5 alpha-pregnane-3,20-dione and [3H]3 beta-hydroxy-5 alpha-pregnan-20-one, were isolated and characterized employing a combination of chromatographic techniques, derivative formation, and crystallization with authentic [14C]5 alpha-pregnane-3,20-dione and [14C]3 beta-hydroxy-5 alpha-pregnan-20-one to constant 3H:14C ratios. In short-term incubations with placenta homogenates, the apparent pH optimum for the synthesis of 5 alpha-pregnane-3,20-dione was 8.8. There was no evidence for the formation of 5 beta-reduced pregnanes from [3H]progesterone by placental tissue. The in vitro metabolism of [3H]progesterone by minces of five term placentas, during 1 hour incubations at pH 7.4, was studied. The rate of formation of 5 alpha-pregnane-3,20-dione varied from 109 to 197 pmoles/gm placental tissue/hr.

[Growth hormone specific binding sites : characteristics of lactogenic receptors induced by estrogens (author's transl)].

In vitro binding of growth hormone was characterized in rat liver. Microsomal preparations were found more active than membranes purified with an aqueous two-phase polymer system. Binding conducted at 4 degrees C was found optimal after 72 hours of incubation in 5 mM Tris-Maleate buffer pH 6.4 with 25 mM CaCl2. Injecting estrone (25 microgram/100 g B.W.) for one week induced the formation of lactogenic receptors, and increased the specific binding of hGH from 2.8 +/- 1.9 to 22.3 +/- 7.1%. Prolactin and hGH, but not rGH or other pituitary hormones, could displace radioactive hGH. With incubations conducted at 37 degrees C, equilibrium was reached more rapidly but at the cost of a more extensive degradation. The presence of membrane receptors in the medium partly protected the hormone against aggregation and degradation. Scatchard plots were obtained from experiments conducted under optimal conditions and analyzed on computer using a program based on an iterative method. Data indicated that lactogenic receptors possessed a single specific binding site for hGH with a constant (Ka) of 2.18 +/- 0.22 x 10(9) M-1 and a binding capacity of 304 +/- 91 fm/mg proteins.

Purification and properties of four species of lysyl oxidase from bovine aorta.

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

Study of urinary excretion of butobarbitone in man in relation to the percentage of ideal body weight.

1 Thirty-three patients with no evidence of endocrine disease, hepatic or renal insufficiency or sleep disorders, were classified in groups 1 to 4 in order of increasing of percentage of ideal body weight (IBW) respectively: less than 90% of IBW, 90--120%, 120--180%, and greater than 180% of IBW. After oral administration of 200 mg butobarbitone, concentration of the intact drug was measured by gas liquid chromatographic assay in urine samples collected during 72 h and at three times in blood. 2 A highly significant negative relationship was found between the cumulative excretion of butobarbitone with urine and the logarithm of the percentage of IBW. In contrast for a given weight, excretion of the drug with urine was found to be weakly correlated with the diuresis. 3 The cumulative urinary elimination of butobarbitone was significantly different between the groups studied, except of the difference between the group 2 and 3 of the patients. No significant difference was found between the renal clearances of butobarbitone in the four groups of subjects. 4 We conclude that redistribution of butobarbitone into adipose tissues can explain the obtained results and that obesity modifies the pharmacokinetics of the drug.

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.

Regional development of norepinephrine, dopamine-beta-hydroxylase and tyrosine hydroxylase in the rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine.

Neonatal rats were injected subcutaneously with 100 mg/kg 6-hydroxydopamine (6-OHDA), or vehicle, on postnatal days 1, 2 and 3. At several times thereafter, determinations of tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities, and norepinephrine (NE) concentration were made in the parietal cortex, cerebellum and pons-medulla in order to assess the extent of initial noradrenergic degeneration induced, and the rate of any ensuing regeneration. By the day following completion of the treatment (postnatal day 4), degeneration of noradrenergic terminals in the parietal cortex and cerebellum was very extensive, with NE levels and DBH activities reduced by more than 80%, and TOH activities reduced by 50%. In the parietal cortex noradrenergic degeneration remained virtually complete; and 9 and 70 days postnatal NE concentration and DBH and TOH activities were all decreased by more than 90--95%. In the cerebellum a progressive regeneration and apparent sprouting of NE fibers was observed. By postnatal day 9, NE, DBH and TOH in this tissue had all recovered to near control levels, and by day 70 these measures exceeded control levels by 95%, 115% and 50% respectively. In the pons-medulla, the initial effect of 6-OHDA on any of the measured parameters was negligible. By postnatal day 9 an increase in NE concentration was apparent, which increased further by day 70 to surpass the control level by 70%. At this same time DBH activity was increased by only 15% and TOH activity was unchanged. Separate analysis of the rostral half of the pons, which contains the locus coeruleus, revealed that on day 70 NE and DBH levels were increased much more substantially than in the whole pons-medulla, and TOH activity was also significantly elevated. This data indicates that the initial amount of degeneration induced by the 6-OHDA treatment is similar in both the parietal cortex and cerebellum, but regeneration proceeds only in the cerebellum. This suggests that noradrenergic fiber growth and regeneration in each target tissue is under independent regulation, possibly by the individual target neurons themselves.

Methodology for demonstrating sustained efficacy of hypnotics: a comparative study of triazolam and flurazepam.

To test for sustained hypnotic efficacy, triazolam (0.6 mg) or flurazepam (30 mg) was given to chronic insomniac patients for 7 consecutive nights in parallel, double-blind design. Triazolam at this dose was an effective hypnotic by all usual subjective measures and did not produce appreciable hangover. Flurazepam performed similarly. For either drug, comparison of the mean scores for the first 2 nights with that for the last 2 nights for any of the parameters did not reveal any significant difference. Thus, both triazolam and flurazepam showed sustained efficacy for 1 week at these doses. Some interesting theoretical and practical questions about the measurement of sustained efficacy of hypnotics in situations of repetitive dosing were addressed by the study. While a placebo control is desirable, the results obtained may be uninterpretable. An acute-care hospital setting may not be the ideal setting for doing such studies. There were indications from the study that the first-night results in a hypnotic clinical trial may be atypical.

Solution conformation of glycosaminoglycans: assignment of the 300-MHz 1H-magnetic resonance spectra of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate, and investigation of an alkali-induced conformation change.

Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl beta-D-glucopyranosiduronate, methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution. As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter-residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H-2 of the glucuronate residue, and the CH3 and H-2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts. Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.

Release of gastric inhibitory polypeptide (GIP) by intraduodenal acidification in rats and humans and abolishment of the incretin effect of acid by GIP-antiserum in rats.

Intraduodenal infusion of 0.05-0.5 N hydrochloric acid dose-dependently increases serum levels of immunoreactive gastric inhibitory polypeptide (GIP) in rats. Immunoreactive GIP released by duodenal acidification is biologically active because it augments the glucose-induced release of immunoreactive insulin (IRI). This augmentation of glucose-induced IRI release by intraduodenal acid can be abolished for 30 min by simultaneous intravenous infusion of GIP-antiserum. From this it is concluded that the initial capacity to augment the glucose-induced insulin release (incretin activity) of hydrochloric acid is due to its ability to release GIP. Later on, other gut factors with incretin activity might be released by hydrochloric acid. Also, in humans, intraduodenal infusion of 0.1 N hydrochloric acid releases GIP without changing serum levels of glucose or insulin. The GIP release is a direct effect of intraduodenal acid and is not mediated via secretin release. Injection of secretin in supraphysiologic doses does not change serum levels of immunoreactive GIP. However, such secretin injections induce a short-term insulin release and a decrease in serum glucose concentration.

Attachment of bacteria to exfoliated cells from the urogenital tract.

To establish urogenital infections, organisms must adhere to the mucosal lining. A differential adherence capacity among various bacterial species was observed when exfoliated urethral and urothelial cells were tested in an in vitro system. No difference in the adherence capacity of a particular species was observed when tested with exfoliated cells obtained from voided urine from different healthy individuals of the same sex. Escherichia coli harvested directly from urine specimens of patients with significant bacteriuria showed a significantly higher capacity to adhere than when obtained from the primary isolation plate. Staphylococcus saprophyticus adhered significantly better to urothelial cells than did Staphylococcus epidermidis. Adherence did not differ when the tests were performed in ultrafiltrated, infected and noninfected urine. Variations of the osmolality did not influence the adherence rate of E. coli. Gonococci showed an increased capacity to adhere when tested in urine of increasing acidity. Gonococci producing T1 colonies adhered by significantly higher numbers per cell than such bacteria producing T4 colonies.

Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.

It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).

Oxidation of horseradish peroxidase compound II to compound I.

In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.

Characterization of the molecular defect in infantile and adult acid alpha-glucosidase deficiency fibroblasts.

Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.

Resistance to the phosphaturic and calcemic actions of parathyroid hormone during phosphate depletion. Prevention by 1,25-dihydroxyvitamin D3.

Recent observations indicate that in thyroparathyroidectomized (TPTX) rats fed a low (0.2 g/100 g) phosphorus diet, the tubular phosphaturic response to parathyroid hormone (PTH) remains markedly blunted even when it is assessed at normal or high plasma concentration and filtered load of inorganic phosphate (Pi). Because 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] decreases the tubular capacity to reabsorb Pi when chronically administered to TPTX rats, we have studied whether this vitamin D(3) metabolite could specifically increase the phosphaturic response to PTH in phosphate-deprived animals. The results show that in Vitamin D-replete TPTX rats fed a low (0.2 g/100 g) phosphorus diet, 1,25(OH)(2)D(3) (2 x 13 pmol/d i.p. for 7 d) markedly enhanced the acute tubular phosphaturic response to PTH (2.5 IU/h i.v.) without affecting the action of the peptide hormone on Ca reabsorption and cyclic-3',5'-AMP excretion. The influence of 1,25(OH)(2)D(3) on the phosphaturic response to PTH could not be ascribed to an increased plasma concentration and(or) filtered load of Pi during the administration of the peptide hormone. However, it could be, at least in part, related to the elevation in the basal level of plasma Pi which was observed in the 1,25(OH)(2)D(3)-treated animals. The results also indicate that 1,25(OH)(2)D(3) significantly enhanced the calcemic response to PTH, which was blunted in these conditions of phosphate deprivation. Unlike 1,25-(OH)(2)D(3), 25-hydroxyvitamin D(3) did not unmask the phosphaturic effect of PTH in phosphate-depleted animals, even when given in doses 100 times larger. Thus, 1,25(OH)(2)D(3) displays a selective and powerful activity in preventing the occurrence of tubular resistance to the phosphaturic action of PTH during Pi depletion. This finding suggests the existence of an important interaction between dietary Pi, 1,25(OH)(2)D(3), and PTH in the homeostasis of phosphate.

Factors affecting the incidence and anti-salmonella activity of the anaerobic caecal flora of the young chick.

Thirty-two different types of anaerobic bacteria isolated from chickens have been tested for anti-salmonella activity in vitro. Under the conditions of the test only Bacteroides hypermegas and a Bifidobacterium sp. were shown to inhibit the salmonellas and this was attributed to the production of volatile fatty acids (VFA's) coupled with a low pH. When these organisms were tested in newly hatched chicks no inhibition of S. typhimurium occurred. Possible explanations for this observation are considered. The pH value and concentration of VFA's in the caecal material were determined in chicks from 0-84 days. In vitro tests with S. typhimurium indicated that, whilst the organism would be able to multiply at the pH and concentration of VFA's found during the first few days after hatching, the rapid increase in VFA concentration during the first 21 days would make this increasingly difficult. The significance of the developing caecal flora in relation to VFA production and pH is discussed. Because certain feed additives are known to influence the carriage of salmonellas, the sensitivity of various caecal anaerobes to these compounds was determined in vitro, generally at 1, 10 and 100 microgram/ml. The additives tested included flavomycin, furazolidone, nitrovin, tetracyline, tylosin, sulphaquinoxaline, virginiamycin and zinc bacitracin. All the organisms tested were inhibited by 100 microgram/ml furazolidone; none were inhibited by 500 microgram/ml sulphaquinoxaline. Changes occurring in the VFA concentration, pH value and microflora of the caeca of chicks fed for 49 days or longer on a normal starter diet or the same diet containing 10 or 100 mg/kg nitrovin have been compared. When the chicks were fed on the diet containing 100 mg/kg nitrovin, the Gram-negative non-sporing anaerobes were eliminated as a significant part of the caecal flora. However, the VFA concentration combined with a low pH in chicks from 2 weeks onwards was still sufficient to inhibit salmonella multiplication. Other possibly interrelated factors which might lead to an increased salmonella carrier rate in the nitrovin-treated chickens are discussed.

Metabolism of niacin and niacinamide in perfused rat intestine.

The metabolism of [14C]nicotinic acid and [14C]nicotinamide by perfused rat intestine was studied by analyzing the 14C-products formed at various time intervals after these substrates were administered intravascularly or intralumenally. Intermediates in the Preiss-Handler pathway contained isotope when [14C]nicotinic acid was administered by either route. Large amounts of isotope in niacinamide and some in nicotinamide mononucleotide (NMN) were also found indicating substantial NAD-glycohydrolase activity in this intestinal preparation. The products from [14C]nicotinamide also included the metabolites of nicotinic acid, due to deamidation of the substrate in the lumen when the exposure was prolonged. In short term studies the amide was absorbed rapidly by simple diffusion with little hydrolysis to nicotinic acid. The primary labeled form found in the perfusate when [14C]nicotinic acid was administered via the lumen for both recirculating and one pass perfusions was unchanged nicotinic acid. The primary forms found in the perfusate when the amide was given were both the amide and the acid for both one pass and for recirculating experiments. The rapid transport of nicotinamide from the lumen to the perfusate and the lack of 14C-metabolites of nicotinic acid in the intestinal tissue following intralumenal injection of [14C]nicotinamide in the living animal suggest that deamidation in the digestive tract is not a major fate of physiological quantities of niacinamide.

Pentobarbitone and skeletal muscle contractions: on the interaction with the effect elicited by the beta-adrenoceptor agonist, terbutaline.

The soleus, a slow-contracting muscle, and the extensor digitorum longus (EDL), a fast-contracting muscle from guinea-pig were prepared for isometric recording in vitro. Subtetanic contractions were evoked by transmural field-stimulation. Pentobarbitone increased the force of contraction in both muscles. In the soleus it shifted the stimulation frequency-response curve to the left. Terbutaline caused a decrease in the force of subtetanic contractions of the soleus, an effect which was dependent on the stimulation frequency. In the presence of pentobarbitone, the stimulation frequency had to be lowered by about 2 HZ in order to maintain the optimum response to terbutaline. The EDL responded to terbutaline with an increased force of contraction. In this case the stimulation frequency was less critical and the effects were the same in the presence and in the absence of pentobarbitone. Experiments with alpha-chloralose yielded results similar to those obtained with pentobarbitone.

Dual action of ouabain on transmitter release at neuromuscular junctions of the frog.

Ouabain increased both spontaneous and evoked transmitter release in Mg++-treated frog neuromuscular junctions. This action developed as a two-step process which affected both miniature end-plate potential (m.e.p.p.) frequency and the binomial distribution of e.p.p.s. During the first part of its action, which lasts for approximately 60 min, ouabain (10(-5) M) increased the m.e.p.p. frequency following a saturable process. The increase in m.e.p.p. frequency was blocked by tetrodotoxin (15 nM). The quantal parameters of release, m and n, showed a significant increase but the parameter p was unaffected. Since the same changes in the binomial parameters were observed in Mg++-treated junctions exposed to low [Na+]0 in the absence of ouabain, it can be concluded that Na+ concentration played an important role in the increase of transmitter release. After 60 min in ouabain (10(-5) M) m.e.p.p. frequency increased by an exponential process. The binomial parameters of transmitter release, m and p, increased while n remained unchanged. This action was not influenced by TTX pretreatment nor was it reproduced by decreasing [Na+]0. The mechanism responsible for this action seems to be the Ca++- releasing effect of ouabain from the cytoplasmic sequestering sites.

The nasopharyngeal culture in acute otitis media. A reappraisal of its usefulness.

Simultaneous cultures of the nasopharynx and middle ear exudate (obtained by tympanocentesis) were obtained from 225 children (mean age, 34 months; median age, 41 months) with suppurative otitis media. A 72% prediction rate for middle ear pathogens was obtained by examining the nasopharyngeal cultures after the strict observance of two essential prerequisites: (1) the nasopharyngeal culture was immediately plated on appropriate solid agar and (2) a semiquantitative method for bacterial enumeration was employed in the reading of the nasopharyngeal culture plates. The technique was most valuable where 2+ (greater than 25% up to 50% of total number of colonies was a single pathogen) or greater of a single pathogen was recovered from the nasopharynx. In only one instance, the semiquantitative nasopharyngeal culture incorrectly predicted the middle ear pathogen if one was recovered. Quantitative nasopharyngeal cultures were particularly useful in predicting the presence of ampicillin-resistant Haemophilus influenzae and group A streptococci as causative agents in otitis media.

[Panarteritis nodosa with positive Australia antigen (author's transl)].

A case of panarteritis nodosa with positive Australia antigen is presented. Panarteritis appeared following serum hepatitis and caused arthromyalgia, abdominal pain, prolonged fever of unknown origin, peripheral polyneuropathy, blood hypertension, and renal insufficiency. A muscular biopsy showed atrophy due to denervation and necrotizing arteritis in various stages causing serious damage to the arteries. Abdominal arteriography clearly demonstrated the existence of aneurismal dilations in the liver, pancreas, and kidneys. The angiographic findings in panarteritis nodose are discussed with special reference to the aneurysms localized in several organs. Their situation is described in detail; it is usually abdominal and more specifically intrarenal. The fact that they occur in a high percentage of cases is helpful when establishing the diagnosis. Lastly, the role of Australia antigen in the development of panarteritis nodose is discussed. It stimulates an immune response and the production of circulating immunocomplexes which are depostied on the vascular walls with complement fixation and damage to the blood vessels. The possibility that other viral agents may be present in the various types of necrotizing vasculitis in humans is commented on.

[The treatment of acid-base imbalance in the intensive care of respiratory diseases].

After giving an outline of pneumogenic respiratory insufficiency, signally that deriving from chronic obstructive bronchopulmonary disease, the Authors describe the intensive care of respiratory insufficiency, first from the anesthesiologist's point of view and then in a broader medical sense. In regard to the latter, the Authors emphasize the importance of material equipment and staff training and teamwork; they also list a number of possible iatrogenic disorders in intensive care. Next they discuss medical aids and more specifically the machinery designed to assist respiration, such as pulmonary ventilators and the "iron lung", as implements that can be used to advantage in medical wards. Then they describe the elements to be used for a correct assessmnet of the severity of respiratory insufficiency, under the following subheadings:--state of coma, if present;--state of acid-base balance, oxemia, and water and electrolyte balance;--circulatory compensation or failure;--need for correcting bronchial obstruction. Through several representative examples concerning the medical correction of alterations of CO2, pH, electrolyte composition, and water and blood volumes, they describe the therapeutic measures to be undertaken particularly as regards the metabolic sequels (alkalosis or acidosis) that may occur in the course of treatment. Coming next to intensive care utilizing mechanical devices, they stress the importance of monitoring the parameters of humoral balance during (and even more so, after) said treatment, in view of avoiding the emergence of iatrogenic disturbances such as the reventilation syndrome and the syndrome of post-hypercapnic metabolic alkalosis.

Concurrent daily infection of calves with Fasciola hepatica and Ostertagia ostertagi.

Three groups of calves were infected daily with either 1500 Ostertagia ostertagi larvae, 20 Fasciola hepatica metacercariae, or 1500 O ostertagi plus 20 F hepatica metacercariae. Weekly measurements were taken of calf weight, faecal egg output, plasma concentrations of albumin, plasma activities of sorbitol dehydrogenase, gamma glutamyl transpeptidase and pepsinogen and standard haematological indices. Calves were killed either 10 or 21 weeks after daily infections began. F hepatica infection had little influence on the size and structure of the O ostertagi worm population or vice versa. Mean worm burdens found at 20 weeks in those animals infected with both F hepatica and O ostertagi were 293 flukes and 20,641 nematodes. While this level of infection is similar to that seen in the disease complex in the field, there was no evidence of clinical disease or any difference in weight gain between the groups in this experiment. Factors other than additive worm burdens are obviously important for the expression of disease under field conditions.

[The atypical forms of psychic excitation or hidden excitations (author's transl)].

If the different forms of depression of humour are nowdays well known and easily surrounded and treated, it's for from being the case for the forms of excitation of humour, though they are as numerous. In these forms, indeed, whether they are atypical maniac attacks of hypomania, it may happen that the pathological nature of psychic excitation posses unnoticed, as well for the patient as for his familiars, or it may also happen that the excitation of humour desguises with "masks" suggesting other troubles, mental or not, which bad to delays in the setting of adapted treatments. These "masks" are essentially: -- hysteria and perturbations of character, in the neurosis register; -- delirious aspects, schizophrenical or confusional, in the psychosis register. In these states of hidden excitation, the most difficult thing, nevertheless, is to obtain from the patient a sincere claim for cares, contrarily to what can be noticed in the states of hidden deppression in which the somatical or psychological "complaints" of the patients are very easily exposed to the physician, a generalist as well as a specialist, and the treatments can be searched for.

[Bromazepam in the treatment of somatized psychogenic disorders (author's transl)].

The clinical effects are reported of the benzodiazepine derivative, bromazepam (Lexotanil) in the treatment of psychosomatic disorders in the course of neurotic, psychovegetative, and masked depressive syndromes. The drug was administered orally in 301 patients (178 males, and 123 females). Target symptoms were anxiety, tension, and varied organic dysfunction of psychogenic origin. The optimum daily dosage was three times 3 mg; the duration of treatment ranged from 1 week to 34 months. The effect of treatment was considered excellent in 51.5%, good in 42.5%, moderate in 2%, and absent in 4%. The most responsive target symptoms were psychogenic disorders of the cardiovascular system and of the gastrointestinal tract, as well as anxiety, while no true antidepressive effect was observed. Drug tolerance is excellent. Slight fatigue, vertigo or a mild reduction in psychomotor activity were complained of by about 10% of the patients and usually occurred with daily doses of 18 mg or more, whereas no other side effects were observed. There was no obvious tendency to drug dependence even after after long-term treatment of up to 34 months. Bromazepam appears to be a superior compound to other anxiolytic and psychovegetatively active minor tranquillisers on account of its mild hypnotic action. Its anxiolytic effect causes additional indirect sleep induction in the above-mentioned conditions.

Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.

Enterotoxicity of Aeromonas hydrophila: skin responses and in vivo neutralisation.

Culture filtrates and partially purified enterotoxins of 6 strains of Aeromonas hydrophila isolated from faeces of diarrhoeic and healthy persons, drinking water, sewage and faeces of domestic animals caused induration and increased capillary permeability in skin of adult albino rabbits. The activities were less in crude enterotoxins indicating partial loss during purification. Heat treatment for 30 min at 60 degrees C and 56 degrees C inactivated the induration and permeability effects of culture filtrates and crude enterotoxins respectively. The activities were most pronounced at pH 8.0 showing gradual loss of activities at lower pH values disappearing completely in culture filtrates at 3.0 and in crude toxins at 4.0. The induration and permeability factor activities of crude enterotoxin were neutralised proportionally in vivo with antitoxin raised against the same in rabbits. Suppression effect was more when antitoxin was injected prior to inoculation of toxin. This study demonstrates that the assay of the activity of skin permeability factor being an easier process than loop test, may be used for assay of enterotoxin of A. hydrophila.

The effect of different extraction procedures on two different molecular weight species of serum NSILA and on the carrier protein of small molecular weight NSILA (NSILA-S).

The influence of Dowex-50 adsorption chromatography on the recovery of two different forms of serum NSILA, large and small mol. wt. NSILA, and on the recovery of the binding protein of the small mol. wt. form was studied and compared with another extraction procedure, gel filtration on Sephadex G-50 in 1 M acetic acid. Partially purified NSILA-S is adsorbed to Dowex-50 at pH 6.8. It can be eluted with 20 mM NH4OH and appears unchanged with regard to its biological activity and molecular weight. Adsorption of 125I-labelled NSILA-S to Dowex-50 does not change its binding characteristics to serum. When serum is chromatographed on Sephedex G-50 in 1 M acetic acid, NSILA is obtained in a large and in a small molecular weight form (NSILA-S). After recombination of the small molecular weight NSILA fraction with the "stripped" serum fraction, which contains large mol. wt. NSILA and a specific carrier protein for NSILA-S, re-chromatography of this mixutre on Sephadex G-50 at neutral pH yields NSILA mostly in the void volume. It adsorbs to Dowex-50. After elution from Dowex, acidic gel filtration on Sephadex G-50 results in an elution pattern which is completely different from that of NSILA-S. Adsorption of serum to Dowex-50 results in a dramatic decrease of the NSILA-S binding activity. It is concluded that Dowex-50 adsorption chromatography of serum 1) inactivates most of the serum NSILA-S binding protein 2) leads to the loss of acid dissociable small mol. wt. NSILA (NSILA-S). Therefore, Dowex-50 adsorption chromatography is not suitable for the subsequent determination or further purification of NSILA-S from whole serum.

Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis.

Aortic smooth muscle cell death is an important initial lesion of atherosclerosis. A number of autooxidation products of cholesterol which has been recognized recently has the capability of inducing rabbits' aortic smooth cell death in vitro. Twelve oxidation derivatives of cholesterol, available commercially, were dissolved in small amounts of ethanol, then added to the culture medium at levels not exceeding 0.8%. The medium contained 10% fetal calf's serum which served as an in situ vehicle for the sterols. The degrees of cytotoxicity were graded and measured as percentage of dying and dead cells in the cultures within 24 hr. 25-Hydroxycholesterol and cholesthan-3 beta, 5 alpha, 6 beta-triol, were the most toxic compounds among the sterols tested. When these oxidation derivatives of cholesterol were added to these cultured cells, they significantly depressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a regulatory enzyme of cholesterol biosynthesis (up to 83% inhibition by 25 hydroxycholesterol at a 3 microgram/ml concentration in culture medium) but the sequence of degree of inhibition was not exactly correlated with that of cytotoxicity. Various mechanisms are speculated. Purified cholesterol showed no cytotoxic effect and minimal inhibition of cholesterol biosynthesis.

Effects of some autonomic drugs on duodenal smooth muscle.

Longitudinal muscle strips (LMS) and circular muscle strips (CMS), 2 mm wide and 1.5--2 cm long, from opossum duodenum were exposed to some autonomic agonists. The cholinergic agonists, acetylcholine, carbachol, methacholine, and bethanechol stimulated only tonic contractions in LMS and tonic followed by phasic contractions in CMS. These effects were abolished by atropine 10(-6) M. The ED50S of all cholinergic agonists for LMS were significantly lower than for CMS. Norepinephrine caused initial contraction (abolished by phenoxybenzamine, 10(-4) M), followed by relaxation (abolished by propranolol, 10(-5) M), and isopropylnorepinephrine caused relaxation (abolished by propranolol, 10(-5) M) in both layers. There were no differences in relative potencies for adrenergic agonists between the layers. Tetrodotoxin did not affect the response to adrenergic agonists. Thus, the potency of cholinergic agonists is greater in longitudinal than in circular muscle, and the layers respond differently to cholinergic agonists. The alpha-adrenergic receptors mediate contraction and beta-adrenergic receptors mediate relaxation on the duodenal smooth muscle.

Effects of acidosis on mechanical function and Ca2+ exchange in rabbit myocardium.

The effects of acidosis on myocardial function and calcium exchange have been studied in the isolated but arterially perfused interventricular septum of the rabbit. Temperature was 28 degrees C and stimulation rate 48 beats/min. Acidosis was induced either by increase of the perfusate PCO2 (pH reduced from 7.35 to 6.68) or by decrease of the bicarbonate-chloride ratio (pH 7.35 to 6.72). The effect on calcium efflux was assessed by introduction of acidosis at different times during the washout of 45Ca2+ from the muscle. The uptake of 47Ca2+ was recorded directly with a NaI crystal and counter. An increase of perfusate PCO2 caused a rapid fall in developed tension. The efflux of slowly exchanging 45Ca2+ and the uptake of 47Ca2+ were inhibited. There was no rapid displacement of calcium from the muscle. Decrease of the bicarbonate-chloride ratio caused a slower fall of developed tension and neither the efflux nor uptake of calcium were altered. These results suggest that developed tension and calcium exchange in the myocardium are more responsive to acidosis within the cell or cell membrane than to extracellular acidosis.

[Hemodynamic and metabolic effects of barbiturates on the human central nervous system].

Anoxic cerebral ischemia results in an almost instantaneous stoppage of the EEG, and a depletion of the cerebral energy reserves with anaerobic orientation. The experimental work, especially of Safar and Michenfelder, have proven the significant cerebral protection under barbituric hypometabolism. The cerebral impacts of barbiturates in the animal, in vitro and in vivo, are multiple: decrease in glycolysis, energetic stabilisation, a blocking of the adenylcyclase activity, membrane stabilisation by trapping of the free radicals or by an increase of the cerebral osmolarity, ect... In man, the metabolic and the hemodynamic data, of which there is little, have only confirmed the reports of metabolic and circulatory depression made by Himwich as early as 1947. The use of barbiturates in postischemic protection poses a problem of their specificity. For an equivalent cerebral metabolic depression a protective effect could not be proven with the tilisation of halogens nor with the morphines... Other narcotics (Alfatésine, Gamma OE) also depress the CNS, but their protective effects are unknown. MICHENFELDER attributes the barbituric protection to the "functional neuron" block without interference at the metabolic level of cellular life. The specificity of barbiturates in this block has yet to be proven. In the post-ischemic resuscitation, Safar insists, and rightly so, on the urgency to correct the cerebral hypoperfusion with a parallel barbituric induction. If there is no "specificity" in the narcotic hypometabolism, it would be logically imperative to use that which provides the best protection, in the shortest, delay, and with the least systemic depression.

[Bacteriological studies of peritonitis].

Bacterial infections of the peritoneum may be primary (pneumococcal peritonitis of childhood). These are rare, as are Streptococcal and Staphylococcal peritonitis in the neonate, following umbilical or tegumental infections. The great majority of cases of peritonitis are secondary to an adjacent inflammation, a perforation of the gastro-intestinal tract, or trauma, which may be surgical in origin. The microbial flora involved is generally mixed, with a high proportion of anaerobic organisms, reflecting the composition of the intestinal flora, and in particular, that of the colon. Infections due to a single organisms are less common, and are seen principally in superinfected ascites, high perforations, or following preoperative antibiotic therapy. The treatment of peritonitis is thus dependent upon the microbial flora present. In mixed infections, broad spectrum antibiotic therapy (amino-glycosides + beta lactamines) is necessary, combined with a b-nitro-ionidazole derivative in view of the frequent presence of Bacteroides fragilis. When the infection is due to one or a limited number of organisms, only the antibiotic sensitivy will provide useful indications. The dangers of preoperative "sterilization", which leads only to the selection of multiresistant strains, is emphasized. Such sterilization is thus to be proscribed in the prevention of peritonitis. Antibiotic cover, on the other hand, is still indicated.

Changes in hydromineral metabolism in diarrhoeic rabbits. 2. Study of the modifications of electrolyte metabolism.

The study of the equilibrium of the main electrolytes (Na+, K+, Cl-), in plasma and intestinal content and the evolution of the partial plasma (Na+, K+, Cl-) in the young rabbit suffering from diarrhoea revealed disturbances of the mineral metabolism. Mineral losses were lower in sick animals than in controls. When the reduction of ingested matter was taken into account, the ion balance showed a deficit. The apparent coefficient of digestive utlization (aCDU) of the electrolytes Na+ and K+ dropped, although the reabsorption of these ions in the last segments of the digestive tract was generally good. The pH remained normal and the proteinaemia fell, suggesting a haemodilution. This was backed up by the presence of a hyponatraemia and a hypochloraemia, but did not account for the large drop in kaliemia. On the other hand, the plasma osmotic pressure increased, very probably in association with a high uraemia. It would therefore seem that the pathogenesis of diarrhoea in the young rabbit differed from that of other known examples, such as diarrhoea in the calf and in the infant. In the young rabbit, the pathogenesis of diarrhoea resulting from coccidiosis would seem to be, as much a result of a nutritional deficiency in particular of potassium, as of a malfunctioning of the digestive tract.

Blood levels of neuroleptic drugs in nonresponding chronic schizophrenic patients.

Blood levels of butaperazine were measured in schizophrenic patients who were chronic nonresponders to their psychotropic medication. The blood levels were compared with those in patients who had shown a better clinical response to this neuroleptic. Nonresponders had two to seven times lower levels of butaperazine in plasma and RBCs after a single dose or chronic dosing. Some of the patients later treated with thioridazine or haloperidol had lower plasma levels of these neuroleptics also. No significant differences were found between nonresponders and relative responders in either the alpha- or beta-phase half-life of butaperazine in plasma and RBCs after administration of a single dose of the drug. Butaperazine and thioridazine levels were not related to previously administered amounts of neuroleptic drugs. These findings do not support the hypothesis that low blood levels are the result of faster systemic metabolism of the drug after it reaches the central circulation. Our results suggest that low blood levels of neuroleptics may be one important factor in the poor clinical response of some chronic schizophrenic patients.

Subendocardial protection during cardiopulmonary bypass. Its use with methylprednisolone and glucose-insulin-potassium.

Fifteen adult dogs were equally divided into three groups: group 1 (control) received no medication before or after cardiopulmonary bypass (CPB), group 2 was administered glucose-insulin-potassium (GIK) according to Maroko's protocol before, during, and after CPB, whereas group 3 was treated with methylprednisolone (30 mg/kg) 30 minutes prior to and near the end of CPB. Moderately severe subendocardial hemorrhage was present in 80% of group 1, 50% of group 2, and only 20% of group 3 ventricles. Endocardial viability ratios after CPB were greater than 0.75 in 20% of group 1, 80% of group 2, and 100% of group 3 animals. Both GIK and steroids improved subendocardial perfusion after anoxic arrest and CPB compared with controls. However, steroid-treated dogs exhibited less endocardial hemorrhage, improved contractility, and more rapid return to baseline conditions than either GIK or control animals. These data suggests that steroids could be considered whenever normothermic anoxic cardiac arrest is used for cardiac operations.

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.

Pharmacological studies on CS-430, a new psychotropic agent.

10-Bromo-11b-(2-fluorophenyl)-2,3,7,11b-tetrahydrooxazolo[3,2-d][1,4]benzodiazepin-6(5H)-one (CS-430) is a new psychotropic drug and has the following properties. The compound showed sleep-inducing effect at both 0.2 and 5 mg/kg (p.o.) in cynomolgus monkeys or at 3 mg/kg (p.o.) in rats. CS-430 showed selective conflict attenuating in rats or anticonvulsant effects in mice. Furthermore, CS-430 blocked non-discriminated (Sidman) avoidance response without severe impairment of the motor function but not discriminated (shuttle-box) avoidance response in rats. CS-430 also blocked selectively electroshock-induced fighting behavior of mice but not isolation-induced fighting behavior of mice and muricidal behavior induced by ablation of the olfactory bulbs. In addition, CS-430 produced specific potentiation of chlorprothixene-induced sleep and particular potentiation of thiopental sleep in mice. These effects of CS-430 were similar to those of nitrazepam, but differed from those of phenobarbital. The effect of CS-430 on the motor function was roughly 1/2 as potent as that of nitrazepam in the various tests.

In vitro studies on GABA release.

1 Recent studies have demonstrated growing evidence for a primary action of the benzodiazepines on gabaminergic neurones which induces a facilitation of gamma-aminobutyric acid (GABA)-mediated neurotransmission. As enhancement of GABA release has been suggested to account for their activation of GABA mechanisms, the effect of diazepam and clobazam, and of several other psychotropic drugs, on stimulated GABA release have been studied. 2 Using rat brain cortex slices saturated with [3H]-GABA, the electrically stimulated overflow of GABA is reduced in a concentration-dependent manner in the presence of both diazepam and clobazam. 3 The benzodiazepine-induced reduction in GABA overflow during electrical stimulation is antagonized by the GABA receptor blocker bicuculline, whereas bicuculline alone at 10(-6) M concentration does not change the overflow. 4 Among some other centrally active drugs tested, hexobarbitone and the 'second messenger; cyclic GMP also induce a significant but less marked reduction in GABA release. 5 A schematic model of a central gabaminergic synapse is proposed, which may explain the benzodiazepine effects on stimulated GABA release by suggesting an inhibitory feedback control of transmitter release mediated by presynaptic GABA receptors ('autoreceptors').

Differential effects of the 1,4 and 1,5 benzodiazepines on performance in healthy man.

1 The immediate and residual effects on performance of benzodiazepines differ, and the differences are important in the use of these drugs. 2. Diazepam and its hydroxylated metabolites, temazepam and oxazepam, each possess hypnotic activity, and have effects on performance limited to the sleep period. The demethylated metabolite of diazepam, nordiazepam, and its precursor, potassium clorazepate, which also possess hypnotic activity, have a longer duration of action, although the next day anxiolytic effect is accompanied by only minimal effects on performance. The 1.5 benzodiazepine, clobazam, seems to have minimal immediate effects on performance. 3 Diazepam and its hydroxylated metabolites, temazepam and oxazepam, would be useful in the management of insomnia without psychopathology in those cases in which residual effects on performance must be avoided. Nordiazepam and potassium clorazepate would be appropriate for insomnia secondary to day-time anxiety, and clobazam may be useful as a day-time anxiolytic. 4 It is emphasized that more work needs to be carried out on the effects of anxiolytics on performance before one can be certain that ingestion during the day would be without any deleterious effects on skilled work.

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.

[Effect of sodium dodecyl sulfate on biological membranes].

Effect of sodium dodecyl sulfate (SDS) on yeast cells resulting in the cellular proteins release from the cells was examined. The influence of pH, ionic strength, the agent concentration and the cells lipids content on the proteins extraction degree was studied. It is assumed that the proteins release is due to the plasma membrane solubilization under the surfactant treatment. It is established that the mechanism of the monimer--membrane interaction and that of the micelle--membrane interaction are likely to be different. It is shown that micelle-like surfactant--protein complexes can solubilize the membrane as well as the SDS micellies. It is found an increase of the ionic strength decreases the efficiency of the SDS action probably due to an increase of the micelle volume. It is assumed that the efficiency of the SDS action on the cell is limited particularly by the ratio of cell wall pere size to the size of micelles formed by the agent in the solution. It is shown also that removal of the largest portion of the lipids from the yeast cell favours an increase in the efficiency of the SDS extractive action at the agent concentrations larger than the critical micelle concentration value probably due to the corresponding decrease of the amount of the cellular components which must be solubilized by the surfactant micelles.

Sensitivity of mouse vas deferens to neurotransmitters: changes after morphine treatment.

1. The pharmacological responses of the isolated vas deferens of the mouse were investigated after acute and chronic treatment with morphine. 2. The addition of morphine to the bath did not alter the responses of the vas deferens to exogenous noradrenaline, adrenaline or dopamine. 3. Low doses of morphine depressed the responses to acetylcholine. Very high concentrations of the opioid (8.5 x 10(-4) M) completely abolished, in about 50% of the preparations, the responses to exogenous acetylcholine, while in the other 50% a potentiation of the responses to low concentrations of acetylcholine was observed. 4. The vas deferens of mice chronically treated with morphine showed increased sensitivity to exogenous noradrenaline, but decreased sensitivity to acetylcholine. 5. A fresh amount of morphine added to the bath enhanced the responses of morphine-tolerant preparations to noradrenaline but not to dopamine or acetylcholine. The specificity of this phenomenon was demonstrated by the use of pentobarbitone instead of the opioid. 6. These results are in agreement with the theory that tolerance could result from a form of disuse supersensitivity.

Selectivity of beta adrenoreceptor antagonist drugs assessed by histamine bronchial provocation.

In a double-blind, within-patient, randomized study, 12 mild asthmatics were given single oral doses of propranolol (80 mg), metoprolol (100 mg), timolol (10 mg), or placebo. Resting heart rate and forced expiratory volume in one sec (FEV1) were measured before and 90 min after treatment. Nonspecific bronchial reactivity was measured by inhaled histamine at 90 min. Following each active drug, resting heart rate changed to a similar extent and to a greater degree than after placebo (p less than 0.01). Changes in FEV1 were small and not different from those after placebo. In contrast, after each active drug, bronchial reactivity increased more than after placebo. The degree of reactivity with each active drug was similar but the differences from corresponding placebo values were significant (p less than 0.05). We conclude that, in mild asthmatics, nonspecific bronchial reactivity is a more sensitive index of airway effects than resting FEV1. Moreover, in the context of this study, since the beta-blockers were given in doses likely to induce equivalent cardiac beta-blockade, there is no evidence to suggest that any one of them is more "cardioselective" than the others.

The metabolic disposition of norgestrel in female rhesus monkeys.

Following single intragastric doses of d- and dl-[24C]norgestrel (Ng), rhesus monkeys excreted 29.5 +/- 2.0 (SE) and 52.6 +/- 5.4% of the administered radioactivity in urine. Fecal excretion accounted for 57.1 +/- 4.0 and 37.2 +/- 4.4%, respectively. Urinary radioactivity was separated into neutral, acidic, and conjugated fractions. The neutral and conjugated fractions contained Ng; 2 alpha, 16 alpha- and 16 beta-hydroxy-Ng; 3 alpha,5 beta-tetrahydro-Ng and 16 beta-hydroxy-3 alpha,5 beta-tetrahydro-Ng and their glucuronides. However, the bulk of the radioactivity in these fractions was associated with more polar metabolites. The acidic fraction contained unstable metabolies which lose 14CO2 at pH values below 5. The most abundant of these metabolites was isolated as its stable methyl ester. The structure of a 13-ethyl-D-homogon-4-ene-3,17 alpha-dione-17 carboxylic acid is proposed for the metabolite which decomposes to 13-ethyl-D-homogon-4-ene-3,17 alpha-dione [D-homo-G]. The probable mechanism of the metabolite's formation is postulated. Quantitative differences in urinary metabolite patterns were observed following the administration of d- and dl-Ng. Ng is the major identified drug-related entity in plasma. The d-Ng concentrations in plasma measured by radioimmunoassay were in good agreement with those determined by fractionation and radiometry. The latter method indicated the presence of D-homo-G and a glucuronide of 3 alpha,5 beta-tetrahydro-Ng following the administration of d- and dl-Ng.

Maturational changes in adrenal xenobiotic metabolism in male and female guinea pigs.

In young (25-day-old) guinea pigs, adrenal and hepatic benzo[a]pyrene (BP) hydroxylase activities were similar but the rates of ethylmorphine (EM) demethylation were greater in adrenals than liver. Between 25 and 50 days of age no sex differences in adrenal or hepatic enzyme activities were demonstrable. The rates of adrenal BP and EM metabolism increased with age in guinea pigs of both sexes; activities reached significantly higher levels in males than females. In contrast, hepatic metabolism of BP and EM declined with maturation and activities were similar in males and females at all ages. Neither microsomal cytochrome P-450 concentrations no NADPH-cytochrome c reductase activities correlated with the maturational changes or sex differences in xenobiotic metabolism. Adrenal microsomal steroid 21-hydroxylase activity did not change significantly with aging and was not sex-dependent. The results indicate that opposite changes occur in adrenal and hepatic xenobiotic metabolism as a function of aging, resulting in substantially greater adrenal than hepatic activity in sexually mature animals. The data also suggest that adrenal microsomal drug and steroid metabolism are independently regulated.

Aminomalonic acid and its congeners as potential in vivo inhibitors of L-asparagine synthetase.

Aminomalonic acid is a strong in vitro inhibitor of L-asparagine synthetase from Leukemia 5178Y/AR and from mouse pancreas; the agent is formally competitive with L-aspartic acid (Ki = 0.0023 M and 0.0015 M for the tumoral and pancreatic enzymes, respectively). Since aminomalonic acid is unstable and inert in vivo as an inhibitor of L-asparagine synthetase, attempts were made to deliver it to the site of its intended action via precursors: the diamide (2-aminomalonamide), the diester (diethylaminomalonate), and the keto acid (ketomalonic acid). Each of these putative 'pro drugs' was shown to be susceptible to metabolism to aminomalonate by mammalian and bacterial enzymes, in vitro. In vivo, aminomalonamide failed to inhibit tumoral L-asparagine synthetase at any time period up to 24 h after its oral or intraperitoneal administration. The diester and keto acid were similarly inactive. However, with specialized techniques it was possible to demonstrate that the diamide significantly inhibited the amidation and/or incorporation of L-aspartic acid into the L-asparaginyl residues of protein. Chemical manipulations of aminomalonic acid aimed at introducing irreversibly reacting functions are warranted.

Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases.

1. NAD(P)+-induced changes in the aggregational state of prepurified NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativaum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55--70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70--95%. It contains a sigle subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38,000/40,000, 38,000/42,000 and 36,000/39,000 respectively. 2. The NAD-specific glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL-6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39,000. 3. The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde-3-phosphate dehydrogenases from higher plants.

One year's experience with the Ypsilon-Y IUD.

In 1975, a two-year study designed to test the new Ypsilon IUD was initiated by the Family Planning Center (Department of Obstetrics and Gynecology, Medical University), Debrecen, Hungary. From November 1975 through September 1976, Ypsilon-Y intrauterine devices (IUDs) were inserted in 300 women who had not previously used IUDs. The control group consisted of 300 Lippes Loop D users, all of whom were also first-time IUD users. The selection was made by randomization. After one year, the relevant rates (pregnancy, expulsion, medical removals) for the study group were: 3.9, 10.4 and 7.8, respectively. The corresponding values for the Lippes Loop D group were: 2.8, 5.7 and 7.8 per 100 women. Although the pregnancy rate for the control population was lower, the difference was not statistically significant. The frequency of expulsion in the Ypsilon-Y users was higher than that of the Lippes Loop D acceptors, but the difference was not statistically significant at the 0.05 level. The medical removal rates were almost the same in both groups. The total number of bleeding, pain and dysmenorrheic complaints in the two groups were also compared and showed that the Ypsilon-Y users did not report complaints as often as did the women wearing the Lippes Loop D. The difference was statistically significant. The relevant termination rates of the Ypsilon-Y were higher than those for the Lippes Loop D; however, the lower frequency of complaints in the study group suggests that the shape of the Y may be more suitable for the uterine cavity than the Loop D.

Physical chemical and technical limitations to intragastric titration.

Intragastric titration, introduced in 1973, has become more popular than in the past and is being applied to the study of gastric secretion under a variety of conditions using NaHCO3 and NaOH as titrant. Physical chemical and technical considerations point to limitations on the conditions under which quantitative results can be obtained. At low end point pH, the limitation relates to volume expansion by secretions and titrant. Errors increase exponentially so that at pH 1.11 a 66% error is incurred and at pH 0.93 a 100% error would result. Neutralization with NaHCO3 poses limitations when CO2 is not effectively removed from the stomach, and even at pH 5.5 errors as large as 20% can be incurred. Errors increase markedly at high end point pH. Use of end point pHs above the pKa' of carbonic acid could lead to large amounts of factitious acid secretion if the intralumental contents are in equilibrium with CO2 entering from the gastric wall. There seems no reason that intragastric titration should not be a useful and quantitative method within the end point pH range of 4.0--4.5. In vivo validation of each experimental condition is needed.

Immunosupression of the primary and secondary immune response by an IgM plasmacytoma (TEPC-183).

Previously we had established that TEPC-183 IgM(K) suppressed the primary immune response (IR) to both the T-dependent antigens 2,4-dinitrophenyl-haemocyanin (DNP-HCY) and T-independent pneumococcal polysaccharides. In the current investigation, the effect of TEPC-183 on an ongoing immune response to SS-III and DNP-HCY was examined. It was found that when TEPC-183 was injected 6 days after the initial antigen injection, at the height of the primary IR, the response was significantly suppressed to SS-III and to the DNP ligand. In addition, suppression of the secondary IR occurred when mice were injected with tumour as late as 35 days after the first antigen injection. Tumour removal lifted the immunosuppression to DNP and the tumour-removed group had a similar number of both direct and indirect anti-DNP-PFC, although HA levels were still reduced. When mice were pretreated with serum from normal mice or serum or ascites from TEPC-183 bearing mice, one day prior to and on the day of antigen injection, the immune response to DNP was reduced by TEPC-183 serum but not by normal mouse serum (NMS), while the anti-SSS-III response was reduced by both NMS and TEPC-183 serum. Thus, NMS selectively suppressed the T-independent response, but only TEPC-183 serum suppressed both types of responses. The suppressive effect of serum on the IR of normal mice indicates a role for soluble regulatory suppressive factors present in the serum of normal and tumour-bearing mice. The data are consistent with the idea that the tumour exerts its effect on the inductive as well as the proliferative phase of the immune response.

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity.

Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigating in vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.

Respiration-linked proton transport, changes in external pH, and membrane energization in cells of Escherichia coli.

The kinetics of respiration-dependent proton efflux and membrane energization have been studied in intact cells of logarithmic-phase Escherichia coli. Parallel measurements of the rate and extent of proton efflux into the external medium (half-time, about 10 s; ratio of H(+) to O, about 0.5) and the oxidation of E. coli cytochrome b (half-time, </=1 s; about 6% oxidized) after a pulse of 5.5 ng-atoms of O indicate that the rate of proton efflux is at least 10 times slower than expected from the time required for the cells to reduce the oxygen added in the pulse. The kinetics of formation and dissipation of the transmembrane electric potential (deltapsi) after an O(2) pulse were estimated from changes in the fluorescence properties of the cell envelope-bound probe N-phenyl-1-naphthylamine. Under anaerobic conditions, a small pulse of oxygen induced a rapid (half-time, </=1 s) partial decrease in the fluorescence intensity of the probe, followed by a slower relaxation of the fluorescence change to the original intensity. The extent of the initial rapid decrease was linearly dependent upon the amount of oxygen added in the pulse (0 to 11 ng-atoms of O per pulse), whereas the rate of the subsequent relaxation was accelerated by the uncoupler p-trifluoromethoxycarbonylcyanidephenylhydrazone and the K(+) ionophore colicin E1. This suggests that the initial fluorescence decrease after an O(2) pulse reflects the energization of the membrane, whereas the relaxation of the fluorescence decrease reflects the subsequent deenergization of the membrane arising from counterion redistributions. The fact that the efflux of H(+) into the external medium after an O(2) pulse was inefficient and much slower (half-time, about 10 s) than the reduction of the added O(2) (half-time, </=1 s) and the energization of the membrane (half-time </=1 s) suggests that some of the protons translocated across the cytoplasmic membrane during a brief pulse of respiratory activity are accumulated in a region of the cell which is not in rapid equilibrium with the external medium.

Activation of rat liver guanylate cyclase by proteolysis.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.

Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

A particulate fraction isolated from human neutrophils by homogenization, then centrifugation at 27,000 g, was demonstrated to generate chemiluminescence. This luminescence required the addition of reduced pyridine nucleotide and was very low in fractions from resting normal cells. Stimulation of neutrophils with opsonized zymosan, phorbol myristate acetate, or ionophore A23187 resulted in marked enhancement of the chemiluminescence measured in subsequently isolated particulate fractions. Stimulation did not boost the luminescence produced by fractions from cells of patients with chronic granulomatous disease. The chemiluminescence of particulate fractions from stimulated neutrophils was linear with increasing protein concentration, had a pH optimum of 7.0, and was higher with NADPH as substrate than with NADH. These results confirm previous studies suggesting that the enzyme system responsible for the respiratory burst in neutrophils is present in this fraction. The particulate fraction was used to examine the nature and origin of neutrophil luminescence by investigating the effect on this phenomenon of certain chemical and enzymatic scavengers of oxygen metabolites. Results suggest that the energy responsible for the luminescence of particulate fractions and, presumably, the intact cell, is derived from more than one oxygen species and that luminescence is a product of the interaction of these species and excitable substrates within the cell.

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.

The effect of thioacetamide on urea cycle enzymes of rat liver.

The urea cycle enzymes, carbamoyl-P-synthetase, ornithine transcarbamylase, arginase and other enzymes related to ammonia metabolism, such as glutamate dehydrogenase, glutamine synthetase and alanine and aspartate aminotransferases,have been studied in thioacetamide-induced liver disease in rats. Urea and ammonia were determined both in serum and in liver extracts. Glutamate and aspartate were determined in liver extracts. There was a marked decrease (in brackets: fraction of control) in carbamoyl-P-synthetase (0.23), ornithine transcarbamylase (0.36) and arginase (0.62). The accumulation of ammonia (3.22) and the decreased urea level (0.80) are well known indications of liver failure. Glutamate dehydrogenase and glutamine synthetase increased respectively to 1.50 and 1.33, and the changes in glutamate and aspartate levels were respectively 1.68 and 0.92; this indicates that the metabolic route: 2-oxoglutarate leads to glutamate leads to glutamine is increased, and thereby compensates for the low rate of urea formation. Aminotransferase activities were respectively 0.43 and 0.25. No significant differences were found in serum aminotransferases, or in the concentrations of ammonia and urea.

An analysis of the inhibitory post-synaptic current in the voltage-clamped crayfish muscle.

1. Inhibitory post-synaptic currents (i.p.c.s) were recorded from the feed-back current through a wire electrode inserted longitudinally into the opener muscle fibre of the claw in the crayfish (Cambarus clarkii). 2. I.p.s.c. rose to its peak in about 3-4 msec and decayed approximately exponentially. The decay time constant at -100 mV was 9.4 msec. 3. The decay time constant decreased as the membrane was hyperpolarized and increased during depolarization. The time constant (tau) depends on voltage (V) according to the relation tau = a exp (AV), with a = 18.6 msec and A = 0.0065 mV-1. Voltage dependence was opposite in direction to that seen at frog end-plates, but in the same direction as that of e.p.s.c. in crayfish muscle. 4. At lower temperatures, the rise and fall times of i.p.s.c.s were prolonged. Q10 for the decay time constant was 2.4 between 22.6 and 12.5 degrees C. 5. When pH was decreased from 7.2 to 5.5, the decay time constant increased by about 50%, with little change in the voltage dependence of the time course. 6. When chloride in the solution was changed to iodide, the decay time constant was increased by a factor of 3, while voltage dependence of the time course was not changed. In bromide solution the decay time constant increased by about 50%. 7. Peak amplitudes of i.p.s.c.s were approximately linear as the membrane was depolarized, but they levelled off as the membrane was hyperpolarized beyond reversal potential. The non-linear I-V relation did not result from inadequate voltage clamping, nor from a change in the inside concentration of chloride. After equilibration with iodide solution the I-V relation was approximately linear. 8. The decay time constant was increased after repetitive nerve stimulation. This prolongation became more pronounced at lower temperatures. 9. The kinetic process of the transmitter action is discussed. It is suggested that the rate limiting process for i.p.s.c. is binding and unbinding of the transmitter to the receptor.

Mechanism of the metabolic acidosis of selective mineralocorticoid deficiency.

The mechanism of generation of metabolic acidosis in selective mineralocorticoid deficiency was investigated in bilaterally adrenalectomized (ADX) rats treated with dexamethasone and in sham-operated (S) rats. ADX rats had significantly lower plasma sodium and bicarbonate concentrations and significantly higher plasma potassium concentrations than S rats did. ADX rats developed negative sodium balance when fed a "zero" sodium diet. The minimum urine pH achieved during sodium sulfate infusion and during ammonium chloride administration was not significantly different between ADX and S rats. Bicarbonate reabsorption and urine minus blood PCO2 gradient were not different between ADX and S rats. For any given urine pH, absolute ammonium excretion was significantly lower in ADX than it was in S rats, both during sodium sulfate infusion and during chronic ammonium chloride administration. Glomerular filtration rate (GFR) was significantly lower in ADX than it was in S rats; ammonium excretion corrected for GFR was not different between the two groups. To determine the role of decreased distal sodium delivery (secondary to decrease in GFR and enhanced proximal sodium reabsorption which resulted from distal sodium chloride wastage) on ammonium excretion, ADX rats were fed 0.9% sodium chloride in an effort to keep body weight constant. Salt-loaded ADX rats had a plasma bicarbonate concentration higher than did S rats. Salt-loading also led to a significant increase in GFR; absolute ammonium excretion was significantly higher than that of other ADX rats with the same degree of acidosis. At comparable levels of GFR, there was no difference in ammonium excretion between ADX and S rats. Ammonium excretion was linearly related to GFR. ADX rats fed a zero potassium diet had significantly greater ammonium excretion than did all other groups of ADX or S rats receiving a normal potassium intake. These data suggest that volume contraction is a major factor responsible for the acidosis of selective mineralocorticoid deficiency.