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A re-examination of curare action at the motor endplate.

Recent evidence indicates that curare, in addition to its competitive' interference with endplate receptors, can block open ionic channels by a 'non-competitive' action on the activated acetylcholine-receptor complex. These findings called for further study of the kinetic behaviour of endplate channels and their modification by curare. Examining impulse-evoked endplate currents and acetylcholine-induced current fluctuations, it is found that the lifetime of the open channel is shortened by relatively high concentrations of curare (greater than 5 micrometer), an effect which shows up most strikingly at hyperpolarized levels of membrane potential (-130 mV and above). No shortening of this kind is observed when a neuromuscular block of equal or greater intensity is produced by a dose of alpha-bungarotoxin. Two other neuromuscular blocking agents, gallamine and pancuronium are shown to have an action on channel kinetics which cannot be explained by competitive receptor binding, but conforms to the hypothesis of rapidly repeated blocking and unblocking of individual ion channels, which had been proposed originally to account for the endplate action of local anaesthetics.

Chemical modification of the surfaces of bacterial cell walls.

The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.

Metronidazole in the prophylaxis and treatment of anaerobic infection.

The influence of prophylactic metronidazole on vaginal carriage rates of anaerobes and the development of postoperative anaerobic infection was studied in 104 women who underwent abdominal hysterectomy. Metronidazole prophylaxis in 54 patients led to a decrease in the anaerobe vaginal carriage rate from 65% pre-operatively to 17% and 28% on the 3rd and 7th postoperative days respectively. In the control group (50 patients) no significant decrease in anaerobe yield was noted, corresponding percentages being 72%, 64%, and 74%. Postoperative infection occurred in 36 patients (28 controls; 8 on prophylactic metronidazole). Wound swabs from all 8 patients in the latter group yielded aerobes, and in 1 patient mixed infection (aerobes/anaerobes) occurred. In 7 of these patients (including the patient with mixed infection), the infection resolved spontaneously, while the 8th patient responded to therapy with metronidazole, kanamycin and ampicillin. In the control patients, 21 cases of postoperative wound infection and 4 of vault infection were seen; wound swabs from patients in the former group yielded aerobes in only 6 cases, and mixed growth of aerobes/anaerobes in 10 cases. Postoperative wound/vault infections in control patients cleared spontaneously in 18 cases and responded to imidazole therapy, with or without ampicillin and kanamycin, in 7 cases.

PCO2 of the proximal tubular fluid and the efferent arteriolar blood in the rat kidney.

Recordings in vivo of the carbon dioxide tension of the proximal tubular fluid and of the efferent arteriolar blood were performed with PCO2 microelectrodes in the rat kidney. The buffer lines of the efferent arteriolar blood and systemic arterial blood were determined with an ultramicro equilibration system and the acid-base status of the systemic arterial blood was measured. The intratubular PCO2 was significantly higher than the PCO2 of the arterial blood, and the PCO2 of the efferent arteriolar blood was significantly lower than that of the arterial blood. The buffer capacity was higher and the bicarbonate concentration slightly lower for the efferent arteriolar blood than for the arterial blood. It is concluded that a PCO2 difference exists across the tubular wall and that the high intratubular PCO2 favours a chemical equilibrium of the carbonic acid-bicarbonate system in the proximal tubular fluid. It is supposed that the slightly lowered bicarbonate concentration in the efferent arteriolar blood is an effect of the glomerular ultrafiltration process.

Protection against fatal endotoxin shock in mice by antihistamines.

Protection against endotoxin shock by antihistamines and similar pharmacologic agents has been reported in the literature. The authors tested the validity of this form of treatment by animal experiments which were conducted in three phases. During the first phase, 10 mice each were treated intravenously with various doses of gram negative endotoxin to determine the dose of endotoxin which would kill 80% of the animals (LD80). This dose was determined to be 36 mg/kg bodyweight. During the second phase, 10 mice each were pretreated with various doses of either diphenhydramine (Benadryl) or of hydroxyzine HCI (Atarax) one hour prior to the administration of the LD80 of endotoxin. It appeared that high doses of diphenhydramine as well as of hydroxyzine were highly fatal to most animals by causing severe convulsions within 3 to 6 hours at doses of 40 or 50 mg/kg. Doses of less than 1 mg/kg appeared to have no protective effect, while doses of 2.5 and of 5 mg/kg, given one hour prior to the LD80 of endotoxin, had some protective value. In the case of diphenhydramine, 60% of the animals survived with 5 mg/kg pretreatment. Hydroxyzine hydrochloride protected 100% of the 10 animals so treated during the initial experiment and 90% during a subsequent experiment, if given 1 hour before the endotoxin. The third phase of this experiment was designed to determine the optimal time at which hydroxyzine needs to be given to protect against fatal endotoxin shock. Given 6 hours before endotoxin, hydroxyzine appeared to protect half of the animals, 1 hour prior to endotoxin, 5 mg/kg of hydroxyzine protected 90% of animals; if given simultaneously, it protected all animals. When hydroxyzine was given 1 hour after endotoxin there was a 70% survival and, if given 3 hours after endotoxin, a 40% survival.

[Assessing functional health and sociability of aged persons: the development and validation of two guttman-scales (author's transl)].

Studies to develop and validate two Guttman-scales are reported, which aim at measuring level of functional health and inclination to engage in social contacts in aged persons. A first scale analysis, based on the answers of 150 old persons, resulted in two, 12-item scales, which according to Guttman's criterion were fully satisfying. In two consecutive studies Guttman-selfratings of two other samples of old people were validated against ratings of experts, who disposed of profound knowledge of the sampled persons (a physician of a nursing home, managers of homes for the aged). A second scale analysis was performed, too. The results, especially the very significant correlations between expert ratings and selfassessments, suggest a broader application of at least the functional health scale. It is proposed to use the scales, if one aims at increasing the fit between individual needs and amount of services provided, and as instruments to control for sample characteristics, if different care programs are evaluated.

The effect of fibrin deposition on the sensitivity of the continuous monitoring pH electrode and on the recorded pH value: an in vitro study.

One of the problems concerning the continuous pH-monitoring technique is whether the relationship between pH measured by the electrode and pH in central and/or capillary fetal blood is constant. To test to what extent a fibrin clot deposited on the pH-electrode influenced the recorded value and the sensitivity of the electrode, the following in vitro study was performed. Fibrin was deposited on the pH-electrode by means of thromboplastin and fibrinogen, or by thromboplastin and whole blood. The deposition of a clot was verified by inspection of the electrode in a microscope. The time for stabilization of the recorded pH-value and the recorded pH-value was measured in standard calibration solutions before and after deposition of the fibrin clot and after decomposition of the fibrin clot by plasmin. FDP was measured in the decomposition solution. From the study it was obvious that the stabilization time of the electrode was considerably influenced by deposition of an "unphysiological" fibrin clot, less so if the clot was deposited by means of whole blood. The recorded pH-value was not influenced.

Telemetered EEG-EOG during psychotic behaviors of schizophrenia.

In an effort to establish correlations between abnormal behaviors characteristic of schizophrenia and simultaneous cerebral electrical activity, EEGs and electro-oculograms (EOGs) were continuously recorded for 2 to 24 hours by radiotelemetry from 40 patients with schizophrenia and 12 normal control subjects. Trained observers recorded specific behavior patterns permitting visual and computer analysis of EEG during hallucinations, stereotypy, catatonia, psychomotor blocking, and other characteristic manifestations of schizophrenia. Electroencephalographic abnormalities consisting of focal slow or spike activity over either temporal region were found in nearly half of the patients so recorded. In contrast to the EEG during ictal episodes of epilepsy, the abnormal wave forms of schizophrenic patients seldom coincided with episodes of blocking, stereotypy, or other abnormal behaviors. Increased extraocular activity or blinking were recorded in a majority of patients, but were not consistently associated with the abnormal behavior or perceptual events.

Histidine residues of zinc ligands in beta-lactamase II.

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0--9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

alpha-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro.

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).

Reactivity of the imino acids formed in the amino acid oxidase reaction.

The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.

How do ionic channel properties depend on the structure of polyene antibiotic molecules?

A study has been made of the properties of ionic channels formed in phospholipid-cholesterol bilayers by polyene antibiotics of various molecular structures. Properties of channels created by natural antibiotics with different structures of the lactone ring (amphotericin B-nystatin-mycoheptin) as well as by some derivatives of amphotericin B modified with respect to the amino and carboxyl groups are compared. Neutralization of one or both charges of the amphotericin B molecule (both by chemical modification and by pH shift) increases the probability of the channel to be in a nonconducting state. An increase of cholesterol concentration in the membrane produces an opposite effect. It is assumed that the electrostatic interaction of the amino group of an antibiotic molecule with the carboxyl group of an adjacent one stabilized the channel. Conductance and selectivity of an open channel are not influenced by changes in the charged groups. These properties strongly depend on the structure of the polar chain of the lactone ring. For example, the appearance of one more carbonyl group in the mycoheptin molecule results in a sharply decreasing anion permeability of channels. An antibiotic concentration which is necessary to observe single channels depends on the polyene chain structure: this is about 10(-7) M for tetraene nystatin and 2.10(-8) M for heptaene amphotericin B an mycoheptin.

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.

Electron microscopic localization of substance P and enkephalin in axon terminals related to dendrites of catecholaminergic neurons.

Morphological and pharmacological data suggest that catecholaminergic neurons receive afferent axons positively labeled for the peptides, substance P and [Met5]-enkephalin. In the present study, electron microscopic immunocytochemistry was used to determine whether a positive reaction for these peptides could be localized to axon terminals forming synapses with catecholaminergic neurons in the locus coeruleus and A2 regions of rat brain. Adjacent sections through these areas were incubated with antiserum to either substance P, [Met5]-enkephalin, or tyrosine hydroxylase, a specific marker for catecholaminergic neurons. The sections were subsequently processes by the peroxidase-antiperoxidase immunocytochemical technique. In both the locus coeruleus and A2 region, tyrosine hydroxylase was localized primarily to perikarya and dendrites of intrinsic neurons; whereas substance P and enkephalin-like immunoreactivity was localized to axons and axon terminals. The axon terminals showing positive reactions for substance P and [Met5]-enkephalin were morphologically similar to each other and to one type of axon terminal which formed synapses with dendrites labeled for tyrosine hydroxylase. This type of axon terminal always formed asymmetric synaptic junctions and contained 3-4 large (75-100 nm) dense vesicles (LDVs) and many small (40-60 nm) clear vesicles (SCVs). The reaction product for substance P and [Met5]-enkephalin was distributed throughout the lumen of the LDVs and formed a rim of labeling around the outer boundaries of the SCVs. These findings demonstrate that substance P and [Met5]-enkephalin-positive reactions are selectively localized to subcellular organelles in axon terminals in the locus coeruleus and A2 region of rat brain. They further suggest that the labeled axon terminals form synapses with dendrites of the catecholaminergic neurons.

Pneumococci resistant to erythromycin.

Susceptibility to erythromycin was determined for all pneumococci isolated in one laboratory from clinical specimens between 1969 and 1977. All 4724 isolates examined prior to October 1973 were susceptible to erythromycin. From October 1973 to December 1977, 64 (0.71%) of 8995 pneumococcus isolates were resistant to erythromycin. The resistant strains were isolated from 38 patients living in six widely separated communities in Alberta. The erythromycin-resistant strains were of nine capsular types, including six that often cause bacteremic disease and five for which resistance to erythromycin has not been reported hitherto. Certain strains of type 33 and of type 15 were highly resistant, the minimum inhibitory concentration (MIC) of erythromycin being 2000 microgram/mL; these strains were also highly resistant to lincomycin and clindamycin. Resistance in strains of other types was much lower, the MIC of erythromycin being 0.6 to 20 microgram/mL, and all but one of these strains were susceptible to lincomycin and clindamycin. All the erythromycin-resistant pneumococci were suspectible to penicillin.

Steroid metabolism in normal mammary gland and in the dimethylbenzanthracene-induced mammary tumor of rats.

After incubation of [4-14C]progesterone with cell-free homogenates of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary tumor of rats, 20 alpha-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol were identified as the metabolites. In normal mammary tissue, however, 4-pregnene-3 alpha-diol was isolated in addition to 5 alpha-reduced, and 3 alpha- and 20 alpha-hydroxy metabolites. When radioactive testosterone was employed as a substrate, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were obtained as the metabolites of the mammary tumor. In the normal mammary gland, only 4-andorstene 3 alpha, 17 beta-diol was formed as its metabolite. Although the enzyme activities relevant to the metabolism varied among the tumor examined, the activity of 20 alpha-hydroxysteroid dehydrogenase in the mammary tumor was significantly lower than that in the normal mammary gland, whereas the activity of 5 alpha-reductase was higher in some of the mammary tumors than in the normal gland. The 5 alpha-reductase activity in the normal mammary gland was mostly localized in the crude microsomal fraction, whereas the same enzyme activity in the tumor was detected in all the organelle fractions. The activities of 20 alpha-hydroxysteroid dehydrogenase and NADPH-linked 3 alpha-hydroxysteroid dehydrogenase were found mainly in the cytosol fractions of the tumor and the normal tissue. The NADH-linked 3 alpha-hydroxysteroid dehydrogenase activity was detected only in the cytosol fraction of the normal mammary gland, but in the tumor studied, the activity of this enzyme was detected in all the subcellular fractions examined.

The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.

UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.

Tumor acceptance modified by passage in hybrids with graft-versus-host reaction.

A graft-versus-host reaction (GVHR) was produced in adult F1 hybrid mice by the injection of 10(8) parental strain spleen cells and 8 days later they were challenged with allogeneic third-party tumor. BALB/c Leydig cell tumor (C4092), C57BL/6 sarcoma (30795), and DBA/1 melanoma (S91) often grew progressively in B6D1F1, CD1F1, B6CF1 or their reciprocal hybrid recipients, respectively, when GVHR had been induced in these animals. Control, without GVHR, hybrids always rejected the tumor. The C4092 tumor was serially transplantable in untreated hybrids after its initial passage in unrelated GVHR-treated mice; the S91 grew in its first passage into untreated B6CF1 mice but thereafter was rejected by these hybrids; while the B6 tumor 30795 grew progressively only in the initial GVHR-treated CD1F1 or reciprocal hybrids. Reduced immunogenicity of tumors resulting from passage in unrelated recipients immunosuppressed in association with a GVHR is comparable to allograft adaptation achieved by such techniques as organ culture pretreatment and presents an additional method for attenuating rejection of allotransplants.

EcoRI activity: enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.

Regulation of lactose catabolism in Streptococcus mutans: purification and regulatory properties of phospho-beta-galactosidase.

Phospho-beta-galactosidase (P-beta-gal), the enzyme which catalyzes the first step in the metabolism of intracellular lactose phosphate, occurred at high specific activity in the cytoplasm in 12 of 13 strains of streptococcus mutans grown on lactose but not other carbon sources. The P-beta-gal from S. mutans SL1 was purified 13-fold using diethylaminoethyl-cellulose ion exchange and agarose A--0.5 M molecular exclusion column chromatography. The molecualr weight of the enzyme was estimated to be 40,000, and its pH optimum was 6.5 in three different buffer systems. P-beta-gal activity was inhibited by Co2+, Zn2+, and Cu2+, but other cations, ethylenediaminetetraacetic acid, orthophosphate, and fluoride had no effect upon enzyme activity. The kinetic response of P-beta-gal to a model substrate, o-nitrophenyl-beta-D-galactopyranoside-6-phosphate, obeyed Michaelis-Menten kinetics, and the Km for this substrate was 0.19 mM. In addition to being under genetic control, P-beta-gal activity was regulated by a number of biologically active metabolites. Enzyme activity was inhibited in a sigmoidal fashion by phosphoenolpyruvate. The M 0.5 V value for phosphoenolpyruvate was 2.8 mM, and the Hill coefficient (n) was 3. In addition, P-beta-gal exhibited strong inhibition by ATP, galactose-6-phosphate, and glucose-6-phosphate. In contrast to inhibition of P-beta-gal activity by phosphoenolpyruvate, the inhibition exerted by ATP, galactose-6-phosphate, and glucose-6-phosphate obeyed classical Michaelis-Menten kinetics; the Ki values for these inhibitors were 0.55, 1.6, and 4.0 mM, respectively.

Inhibition of the interaction between the complement component Clq and immune complexes.

Several groups of compounds were examined for their ability to inhibit in vitro the binding of Clq to insoluble immune complexes. As expected from previous studies, polyinosinic acid, liquoid, sodium pentosan polysulfate, aliphatic diamines and heparin are good inhibitors. This study shows that compounds with much lower toxicity, such as certain amino acids and substances with vitamin B6 activity (pyridoxal-5-phosphate:P5P) were also capable of decreasing the binding of Clq. Using methods of equilibrium dialysis and difference spectra, it was shown that this compound binds to both, Clq und IgG antibody by forming Schiff bases. Clq binds approximately 10 times more P5P when compared to IgG. Immune complexes prepared with IgG antibody modified by P5P and stabilized with sodium borohydride had the same complement-fixing and Clq-binding capacity as normal immune complexes. This suggests that the inhibition of Clq-binding to immune complexes by P5P is due to a modification of lysyl resides in Clq. Collagen and IgG fragments derived from Fc were also found to inhibit Clq binding to immune complexes, but at higher concentrations than the above small-molecular compounds and with a different mode of action.

A quantitative study of the effects of acetylsalicylic acid on spermatogenesis and organs of the rat.

Although the occurrence of prostaglandins in the male reproductive organs is consistent, their physiological role in fertility and reproduction is not known. The influence of acetylsalicylic acid, an inhibitor of prostaglandin synthesis, on the male reproductive tract was investigated in Sprague-Dawley rats of proven fertility. Acetylsalicylic acid dissolved in phosphate buffer was administered once a day at two dosages (300 mg/kg body weight and 150 mg/kg body weight) over a period of 12 days and a period of 6 days. The animals were killed 24 hours after the final treatment and the testes, epididymides, ductus deferens, seminal vesicles, kidneys, and adrenal glands were removed and placed in Bouin's solution. Subsequently, the tissues were cleaned and weighed and the testes were prepared for quantitative study under the light microscope. Organ weights were not significantly altered in the animals that were treated with acetylsalicylic acid. Cell counts indicated that there was a significant increase in the mean number of preleptotene spermatocytes and spermatids in those animals treated with the drug for 6 days at a dose of 150 mg/kg body weight. Treatment at the same dosage for a period of 12 days produced a significant decrease in the mean numbers of preleptotene and pachytene spermatocytes and spermatids. The mean diameter of the seminiferous tubules was also significantly decreased in the latter group of animals. In the group of animals treated with ASA for 12 days at a dose of 300 mg/kg body weight the mean diameter of the seminiferous tubules was significantly increased. No clear conclusion as to the effect of the drug on spermatogenesis or the various organs could be drawn.

Identification and observation of alkyl proton resonances of the amino-terminal residues of bovine neurophysins. Evidence for conformational differences between neurophysin-I and neurophysin-II.

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation of these spectra has allowed identification of the -CH3 proton resonances of the amino-terminal alanine of both proteins and of the -CH3 resonances of methionine-2 of neurophysin-II. The alanine -CH3 resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH3 resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated alpha-amino and an unprotonated side chain carboxyl. The results suggest that differeces in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. 'reporter' signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 A from residue 3 of peptides bound to the strong neurophysin hormone-binding site.

Cellulases released during the germination of Dictyostelium discoideum spores.

Dormant spores of Dictyostelium discoideum contained cellulase at a specific activity of 130 to 140 U/mg of protein; when heat activated, the spores germinated, progressively releasing the cellulase activity into the extracellular medium. The cellulase release was a selective process and resulted in recovery of the cellulase activity at a specific activity of 2,000 U/mg of protein; beta-glucosidase in the spores remained completely associated with the emerging amoebae. Release of the cellulase required heat activation of the spores and occurred during the swelling stage of germination; inhibition of the emergence stage with cycloheximide had no effect on the release of the cellulase. The cellulase activity released consisted of two enzymes whose molecular weights were 136,000 and 69,000. Studies of their pH optima, heat lability, and of their sensitivity to inhibition revealed no distinctive differences between these two proteins. Analysis on diethylaminoethyl-Sephadex columns showed that the higher-molecular-weight protein could be converted into the lower-molecular-weight component in vitro.

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.

Cytochrome c1 complexes.

Cytochrome c1 forms an active complex with cytochrome c as previously reported (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36). It also forms a complex with cytochrome oxidase with heme ratio of 1:1. This cytochrome c1.oxidase complex has been purified by ammonium sulfate fractionation and is stable in media of high ionic strength (greater than 0.1 M) but dissociates as the pH deviates from neutral. The purified cytochrome c1 aggregates to an oligomer, presumably a pentamer. No agent has been found to depolymerize isolated c1 without denaturation. However, in the cytochrome c1.oxidase complex, these two cytochromes apparently were depolymerized to form smaller aggregates, if not monomeric units, as judged by sedimentation behavior. Cytochrome c1 also forms a ternary complex with cytochrome c and oxidase in the heme ratio of 1:1:1. This complex can be prepared by any of the following four methods: (i) c1 + c + oxidase: (ii) c1.c complex + oxidase; (iii) c1 + c.oxidase complex: or (iv) c + c1.oxidase complex. The mode of formation of these complexes is all from pure protein-protein interactions. Cytochrome c1 is also incorporated into phospholipid vesicles and these vesicles show about 200 molecules of phospholipid/cytochrome c1 in terms of heme. The spectrophotometric, circular dichroic, sedimentation behavior and enzymic properties of these complexes have been investigated.

Starting transients in sea urchin sperm flagella.

Live sea urchin spermatozoa were rendered immotile by lowered pH; Triton-extracted spermatozoa were rendered immotile by either lowered pH or by deprivation of ATP. The spermatozoa began to beat after an increase in pH or as ATP was supplied, and the first bends were recorded on ciné film. Triton-extracted spermatozoa deprived of ATP retained a partially formed basal bend which could be either principal or reverse, and which resumed its development and propagation as ATP was supplied. Both live and tritonated flagella straightened at low pH. As the pH was increased, a series of principal bends formed near the base and propagated to the tip. Reverse bends began to develop as the pH continued to increase. The principal and reverse bends thus exhibited different sensitivities to pH, which suggests differences in the mechanisms that produce them. Straight flagella began to move by synchronous sliding all along the flagellum, thus forming principal bends. Flagella that contained a basal bend began to move by primarily metachonous sliding within that bend.

Renal transport and renal metabolism of 2, 4, 5-trichlorophenoxyacetate by the chicken.

In vitro and in vivo renal tubular transport of 2,4,5-trichlorophenoxyacetate (2,4,5-T), a hormone-type herbicide, was studied in the chicken. Renal cortical slices incubated with 14C-labeled 2,4,5-T demonstrated a slice-to-medium ratio of the 14C-label of 26 after 2 hr of incubation. This accumulation was inhibited significantly by probenecid, phenol red, dinitrophenol and iodoacetamide. In vitro metabolism of 2,4,5-T was apparent in the renal slice studies; at least two metabolites were found by electrophoretic analysis. Even though chicken liver slices were able to accumulate 2,4,5-T, the accumulated herbicide was not metabolized. In addition, chicken kidney slices metabolized 2,4,5-T to a much greater extent than did rat or rabbit kidney slices, which produced only negligible amounts of metabolites. When [14C]-2,4,5-T was infused into the renal portal system of the chicken, the 14C-label originally associated with 2,4,5-T was shown to be secreted by the renal tubule. Probenecid inhibited the transport of the 14C-label without affecting the simultaneous transport of tetra-ethylammonium, an organic cation transported by the renal proximal tubule. At the present infusion rate of 2,4,5-T (2.9 X 10(-10) mol/min), the excreted label was associated primarily with an acidic metabolite(s). It can be concluded that the chicken transports 2,4,5-T by a probenecid sensitive mechanism; however, this transport appears to be associated with the metabolism of the herbicide.

Topical mosquito repellents XI: carbamates derived from N,N'-disubstituted diamines.

Carbamates derived from various N,N'-disubstituted diamines were synthesized and evaluated as repellents for Aedes aegypti mosquitoes with an in vitro blood-feeding test system. Several compounds were more effective than diethyltoluamide.

Study of the two pathways for arachidonate oxygenation in blood platelets.

During collagen-induced blood platelet aggregation, arachidonic acid is set free from membrane phospholipids and subsequently converted into 12-hydroxyeicosatetraenoic acid by arachidonate lipoxygenase and into thromboxane A2, 12-hydroxyheptadecatrienoic acid (HETE) and malondialdehyde by cyclooxygenase and thromboxane synthase. Lipoxygenase and cyclooxygenase have optimal activity at neutral to basic pH, while the thromboxane synthase is pH-independent between 5 and 9. These enzymes are membrane-bound. The cyclooxygenase is rapidly inactivated upon membrane disruption by nonionic detergents or phospholipid degradation with phospholipase A2. It was found that platelet phospholipase A2 preferentially splits off fatty acid with four double bonds. Eicosatetraynoic acid was used to investigate the physiological function of the arachidonate lipoxygenase during collagen-induced aggregation of rat blood platelets. This fatty acid is a more efficient inhibitor of lipoxygenase than of cyclooxygenase. At an inhibitor concentration of 0.6 microgram/ml, platelet aggreation, 12-hydroxyeicosatetraenoic acid production as well as 15-hydroxytryptamine release are completely inhibited, while there is an apparent stimulation of the cyclooxygenase. These results indicate that arachidonate lipoxygenase is essential for irreversible blood platelet aggregation.

[Methylviologen photoreduction by chloroplasts].

The condition of methylviologen photoreduction by chloroplasts was investigated. Argon bubbling through the suspension of chloroplasts or degasing in vacuum caused inhibition of methylviologen reduction probably due to the denaturation of chloroplast membranes at the water/air boundary. Adding glycerol or bovine serum albumine or removing oxygen from chloroplast suspension with the aid of the oxygen absorbing-systems preserved the activity of chloroplasts. Methylviologen photoreduction is inhibited by DCMU (10(-7) M) and Tris-buffer treatment and is activated by uncouples. The pH-dependence is similar to that of the Hill reaction. Triton X-100 (0.007%), ethyl ether (2%) and heating up to 42 degrees activated the Hill reaction but inhibited methylviologen reduction. Water molecule probably acts as an initial electron donor in this reaction. It is proposed that the steady level of methylviologen photoreduction is determined by a relationship between the rate of methylviologen electron acceptance and cyclic electron flow short-circuiting photosystem I.

Some factors influencing the neurotoxicity of intrastriatal injections of kainic acid.

Intrastriatal injections of kainic acid are known to destroy striatal neurons including many containing choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Using these enzymes as indices of neuronal loss, the neurotoxicity of small doses of kainic acid was found to be influenced by injection time and volume. It was partly blocked by coinjection of some but not all glutamate antagonists or by prior lesioning of the corticostriatal tract. Other adjuvants, drugs, or lesions tested had little modifying effect, except that changes in the dopaminergic system seemed to increase the toxicity towards cholinergic but not GABAnergic systems. High-affinity glutamate accumulation by neostriatal synaptosomes was significantly increased 1--7 days following kainic acid injections. MAO and acetylcholinesterase activities were depressed in kainic acid-lesioned striata but not nearly as much as were CAT and GAD. An indirect mechanism involving glutamate release and inhibition of reuptake is suggested for kainic acid neurotoxicity.

X-ray diffraction studies of fibers and crystals of deoxygenated sickle cell hemoglobin.

Paracrystalline fibers of deoxygenated sickle hemoglobin in erythrocytes or concentrated solutions exhibit a phase transformation to a fully crystalline state. X-ray diffraction patterns of the fiber and crystallites are similar except in two respects: the equatorial spacings of the fibers suggest that they pack into a square lattice with a = 220 A, whereas those of the crystals can be indexed on the basis of a net of 187 A by 54 A, and the second-order near-meridional reflections are strong on the fiber pattern but weak on that of the crystallites. The crystallites are isomorphous with single crystals grown in polyethylene glycol solution at pH 4.5 whole structure has been determined at near-atomic resolution (Wishner, B.C., Ward, K.B. Lattmen, E.E. & Lowve, W.E. (1975) J. Mol. Biol. 98, 179-194). Double filaments of molecules with an axial repeat of 64 A comprise the basic unit of both the crystal and fiber structures. Each filament of the pair is translated with respect to its neighbor by half a molecular diameter along the fiber axis. The two filaments are held together by contacts made by Val 6beta in the molecules of one strand with hydrophobic side chains of the molecule in the neighboring strand. This interaction is probably the cause of the aggregation of filaments into fibers that leads to the sickling of erythrocytes.

Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides.

After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.

Local development of action potentials in slow muscle fibres after complete or partial denervation.

Pyriformis muscles of Rana temporaria were completely or partially denervated by cutting the sciatic nerve or some of the small nerve branches entering the muscle. One stimulating and one to three recording microelectrodes were inserted along the fibres in order to compare the electrical activity at these points. In an early period following denervation action potentials of variable size and shape could be observed; these action potentials were often composed of two, sometimes of three or four, components. The size of individual components depended on the position of the recording microelectrode. Individual components could occasionally be triggered separately by adjusting the strength of the stimulating current pulse; propagation of these "all or none" responses was absent. In other fibres one component of the action potential could trigger another one several millimetres apart, thus indicating propagation. Conduction velocities were approximately 0.4 m/s. In partially denervated slow fibres, endplate potentials were confined to one lateral segment of the fibres, while the action potential occupied the denervated part of the membrane. The amplitudes of endplate and action potentials varied inversely with distance. Rough estimates of the length constant of the slow fibre membrane were calculated from the spatial decay of action potentials, endplate potentials and hyperpolarizing electrotonic potentials; mean values obtained were 2.5, 4.8 and 7.7 mm respectively. The results suggest that following denervation Na channels are built into discrete areas of the slow fibre membrane and that this process depends on the amount of denervation in individual fibres.

Therapeutic antagonism between anticholinergics and neuroleptics: possible involvement of cholinergic mechanisms in schizophrenia.

We have answered technical criticisms of our work in which anticholinergic agents were added to ongoing neuroleptic treatment in an ABA' research design. The suggested analysis of variance for repeated measures of the three periods is inappropriate because of the expected carryover effects from continuous neuroleptic treatment. The multivariate analysis of various parameters seems unsuitable because homogeneity of covariance cannot be ensured due to the heterogeneity of schizophrenia and the diverse factors represented in the psychopathology measures. We have summarized the results of recent parametric and nonparametric analyses of combined data from our three studies to show that the significant effects clearly pointed to therapeutic antagonism between anticholinergic agents and neuroleptics. We suggest that cholinergic neurons may be part of some crucial discriminative control mechanisms in the brain organization that are ineffective in schizophrenia and lead to a relative overactivity of the opposing catecholaminergic neurons in the midbrain-limbic circuitry which promote repetition of behaviors in goal-directed activity.

[Dietary and drug treatments of calcium nephrolithiasis (author's transl)].

Dietary and drug treatments of calcium nephrolithiasis depend mainly on the mineral composition of renal stones: calcium oxalate, phosphate or mixed stones. The association with an hypercalciuria is an important factor which must be taken into account because oxalates and phosphates precipitate as calcium crystals in case of urinnary oversaturation. Despite many therapies have been proposed, their efficiency seems to be rather small when they are used alone. Usually, it is necessary to act on several factors with a combination of therapeutic methods. Absorptive hypercalciuria are improved with both low calcium diets and inhibitors of calcium absorption. In renal hypercalciuria, the treatment is based on the administration of thiazide diuretics which enhance calcium renal tubular reabsorption. The other therapeutic methods depend on the nature of renal stones: urinary acidification for calcium phosphate; administration of succinimide, oral phosphate or organic phosphonates for calcium oxalate stones; association with purine biosynthesis inhibitors in case of the presence of urates in renal calculi.

Deviated lysis: lysis of unsensitized cells by complement. V. Generation of the activity of low pH or low ionic strength.

In serum exposed to acid pH (6.4), a serum activity was generated which lyzed unsensitized erythrocytes in the presence of EDTA. It was similar to the d.l. activity found following serum activation by inulin (2). In contrast to the d.l. generation by the classical or by the alternative pathway of C activation, the generation of d.l. by acid pH did not require C4 plus C2 or C3 plus factor B resp. It was, thus, not dependent on any hitherto known pathway of C activation. A similar activity appeared when NHS was centrifuged in a sucrose gradient at low ionic strength. Physicochemical alterations of the component proteins which influence their affinity for each other are seen as the basis for the activation of the attack phase of C.

Determination of liver intracellular pH in vivo and its homeostasis in acute acidosis and alkalosis.

An in vivo method is presented for the determination of liver intracellular pH (pHi) using [14C]dimethadione (DMO) in dogs. This method differs from those previously published in that hepatic venous and portal venous blood pH were selected as the extracellular reference pH, and liver blood space corrections are applied to whole liver tissue [14C]DMO activity. Using these corrections, a normal liver pHi of 6.99 +/- 0.03 (SE) was obtained. During acute metabolic acidosis and alkalosis, as well as during acute respiratory acidosis and alkalosis, the liver pHi remained normal; metabolic acidosis was 7.04 +/- 0.04; metabolic alkalosis was 6.92 +/- 0.08; respiratory acidosis was 6.98 +/- 0.04; and respiratory alkalosis was 7.00 +/- 0.10. None of these values was significantly different from normal (P greater than 0.05). Changes in intracellular bicarbonate and lactate appeared to account in part for the observed stability of the liver pHi despite acute manipulations resulting in a range of pH values between 7.09 and 7.63 in arterial blood.

Application of oxygen-enriched aeration in the production of bacitracin by Bacillus licheniformis.

The physiological effects of controlling the dissolved oxygen tension at 0.01, 0.02, and 0.05 atm by the use of oxygen-enriched aeration were investigated during growth and bacitracin production by Bacillus licheniformis ATCC 10716. Up to a 2.35-fold increase in the final antibiotic yield and a 4-fold increase in the rate of bacitracin synthesis were observed in response to O(2)-enriched aeration. The increase in antibiotic production was accompanied by increased respiratory activity and an increase in the specific productivity of the culture from 1.3 to 3.6 g of antibiotic per g of cell mass produced. Oxygen enrichment of the aeration decreased medium carbohydrate uptake and the maximum specific growth rate of B. licheniformis from 0.6 h(-1) to as low as 0.15 h(-1), depending upon the level of enrichment and the conditions of oxygen transfer rate (impeller speed). The response of this culture to O(2) enrichment suggests that this method of controlling the dissolved oxygen tension for antibiotic-producing cultures may simulate conditions that would occur if the carbon source were fed slowly, as is often employed to optimize antibiotic production. Analysis of the biologically active bacitracins produced by B. licheniformis ATCC 10716 suggested that the ratio of biologically active peptides was not changed by O(2) enrichment, nor were any new biologically active compounds formed.

Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody.

Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.

Effects of H1- and H2-receptor blocking agents on histamine-induced bronchoconstriction in non-asthmatic subjects.

1 Two studies have been carried out to investigate the effect of H1- and H2-receptor blocking agents on histamine-induced bronchoconstriction in non-asthmatic subjects. 2 The H2-receptor blocker cimetidine administered orally had no effect on histamine-induced bronchoconstriction on any of the subjects tested. In three of four subjects, the H1-receptor blocker, chlorpheniramine given orally, inhibited the effect of the histamine in the lung. 3 The effects of intravenous chlorpheniramine and cimetidine, both alone and in combination, upon histamine-induced bronchoconstriction, were also studied. Chlorpheniramine inhibited the effect of the histamine and this was significantly dose related. This was not so with cimetidine and there was no evidence that the dose response curve to chlorpheniramine was affected by the additional administration of cimetidine. 4 The results show that histamine-induced bronchoconstriction in non-asthmatic subjects is not mediated by H2-receptors, but it is likely that H1-receptors are involved.

Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase.

Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.

The role of the hypogastric nerve in bladder and urethral activity of the dog.

1. Stimulation of the hypogastric nerves increased the pressure in both the bladder and urethra of anaesthetized female dogs. 2. The responses were reduced but not abolished by the alpha-adrenoceptor antagonist phentolamine, whereas the beta-adrenoceptor antagonist propranolol was either without effect or increased the responses. Atropine, methysergide and hexamethonium were without effect. 3. Close arterial injection of phenylephrine increased and isoprenaline decreased urethral pressure but both produced only a slight increase in bladder pressure. 4. Hypogastric nerve stimulation reduced subsequent responses of the bladder and urethra to pelvic nerve stimulation or to close arterial injection of acetylcholine. Isoprenaline, but not phenylephrine, also had an inhibitory action and 5-hydroxytryptamine enhanced the responses. 5. In the presence of hexamethonium the inhibitory action of isoprenaline still occurred but 5-hydroxytryptamine no longer enhanced the responses, suggesting that 5-hydroxytryptamine acts on the ganglia and isoprenaline acts, at least partially, on smooth muscle. 6. These results suggest that the role of the hypogastric nerves may be to modify inputs to the bladder and urethra as well as to act directly on the smooth muscle.

Assessment of the effects of neonatal subcutaneous 6-hydroxydopamine on noradrenergic and dopaminergic innervation of the cerebral cortex.

Female rats, treated at birth with 6-hydroxydopamine (3 x 100 mg/kg s.c. at 24 h intervals) or vehicle, were subjected at 112 days of age to unilateral electrolytic lesions of the locus coeruleus. Two weeks later regions of the telencephalon, both ipsi- and contralateral to the lesion, were simultaneously assayed for norepinephrine (NE) and dopamine (DA) content, and for tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities. In the vehicle-treated rats the lesion resulted in at least an 80% reduction of NE and DBH on the ipsilateral side, relative to the contralateral side. TOH was reduced to a similar extent only in the parietal cortex and hippocampus. In the prefrontal cortex and cingulate gyrus TOH was decreased by only 31% and 64% respectively; the remainder was interpreted to be associated with projections of the mesocortical dopamine system. From this data it was possible to calculate that the ratio of TOH to DA in dopaminergic terminals is about 10-fold greater than the ratio of TOH to NE in noradrenergic terminals. Neonatal 6-hydroxydopamine treatment resulted in practically total elimination of noradrenergic terminals throughout the telencephalon, and the locus coeruleus lesion had no additional effect. The drug treatment produced no significant change in DA content or in the TOH to DA ratio in the prefrontal cortex and cingulate gyrus, indicating complete sparing of the mesocortical DA projections.

Testicular teratoma in an equine cryptorchid.

An abnormal cryptorchid testicle removed from the abdominal cavity of a 4 year old Thoroughbred stallion is described. The abnormal organ conforms to the requirements of Willis (1960) for a teratoma. The difference between these tumours in man and horses is discussed.

In vitro proliferation of haemopoietic cells in the presence of adherent cell layers. I. Culture conditions and strain dependence.

The culture system, in which a marrow-derived adherent cell population, established in vitro, exerts a long-term promoting influence on proliferation of haemopoietic cells, is reproduced. Essential parameters of the system are investigated; it is confirmed that the system is critically dependent on horse serum, and on the in vitro age of the adherent cell layer. The growth-promoting effect on haemopoietic cells seems to be independent of the number of marrow cells per culture flask initially inoculated into the cultures to establish the adherent cell layer. In vitro established marrow-derived adherent cell layers from RFM (H-2f) and BALB/c (H-2d) mice can promote the long-term proliferation of syngeneic and allogenic haemopoietic cells; haemopoietic marrow cells from C3H (H-2k) cannot be maintained on syngeneic or allogeneic (BALB/c, H-2d) adherent cell layers; adherent cell layers of C3H (H-2k) can maintain haemopoietic cells of the H-2d (BALB/c) genotype. This culture system does not reproduce the in vivo phenomenon of allogeneic resistance. The relevance of these findings to the suggestion that the growth-promoting activity of adherent marrow cells on haemopoietic stem cells in vitro duplicates aspects of the in vivo haemopoietic microenvironment is discussed.

Hepatocellular injury with distinctive mitochondrial changes induced by lergotrile mesylate: a dopaminergic ergot derivative.

Increased serum activities of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) occurred in 12 out of 19 patients with idiopathic parkinsonism when they were treated with the ergot derivative lergotrile at an oral dose varying from 50 to 150 mg daily. Hepatocellular injury was confirmed by microscopic examination of liver biopsies obtained from 3 of these patients when the serum activities of ALT and AST were appreciably elevated. Light microscopy revealed features of mild acute hepatocellular injury, and electron microscopy showed proliferation of the smooth endoplasmic reticulum and apparently unique mitochondrial changes in hepatocytes. This is the first report of pathological changes in the liver associated with the therapeutic use of an ergot derivative. The presence of a potentially reactive cyanide group in the lergotrile molecule could be causally related to the observed hepatocellular injury. It is suggested that serum ALT and AST activities should be monitored carefully when the therapeutic potential of any new ergot derivative is assessed.

Observations on the gubernaculum during descent of the testis.

The mechanisms that influence the descent of the testis are not clearly understood. The gubernaculum is a structure worthy of scrutiny inasmuch as it is conspicuous during descent, but virtually disappears after descent is complete. Early in gestation, the rat gubernacular bulb consists of loose mesenchymal cells that develop into fibrillar cells. These later thicken into rhabdomyoblasts that, near the end of gestation, differentiate into spiral striated muscle bundles, and eventually migrate outward into the abdominal/scrotal wall. The rhabdomyoblasts of the female gubernaculum do not differentiate further but rather undergo fatty degeneration. It is possible that spiral contractions of the attached gubernaculum produce tension on the testis and induce descent. The gubernaculum as the receptor organ for testicular descent may be responsive to local testicular hormones. Likely candidates are testosterone, dihydrotestosterone, or Mullerian Inhibiting Substance. A thorough knowledge of the sequential differentiation of the gubernaculum during embryonic development sets the stage for the study of its response to hormonal manipulation both in vivo and in vitro.

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.

The mechanism of the skeletal muscle myosin ATPase. III. Relationship of the H+ release and the protein absorbance change induced by ATP to the initial Pi burst.

Several phenomena are associated with the binding of ATP to myosin: 1) a fluorescence enhancement, 2) a release of H+, and 3) a protein absorbance change. In the accompanying paper (Chock, S. P., Chock, P. B., and Eisenberg, E. (1979) J. Biol. Chem. 254, 3236-3243), it was demonstrated that the fluorescence enhancement is mainly caused by the hydrolysis of ATP in the initial Pi burst rather than by the conformational change induced by the irreversible binding of ATP. In the present study, the cause of the H+ release and the protein absorbance change were investigated. The results show that like the rate of the fluorescence enhancement the rates of the H+ release and the protein absorbance change level off at high ATP concentration at a much lower rate than the rate of irreversible ATP binding. Furthermore, under all conditions tested, the rates of the H+ release and the protein absorbance change are equal to the rate of the initial Pi burst. Therefore, like the fluorescence enhancement, most of the H+ release and the protein absorbance change are associated with the initial Pi burst rather than the binding of ATP.

Adrenergic modulation of pancreatic A, B, and D cells alpha-Adrenergic suppression and beta-adrenergic stimulation of somatostatin secretion, alpha-adrenergic stimulation of glucagon secretion in the perfused dog pancreas.

The effects of adrenergic substances on pancreatic insular secretions were studied in a completely isolated canine pancreas with exclusion of the duodenum from the perfusion circuit. To ensure adequate blockade, blockers were infused before agonists. A dose range of beta-receptor blockade was tested, and putative alpha-adrenergic effects were confirmed by combined alpha- and beta-adrenergic receptor blockade.beta-Adrenergic agonism (2 ng/ml isoproterenol) induced a mean integrated increase of 79+/-20% in somatostatin secretion, whereas glucagon and insulin secretion were increased by 185+/-45 and 495+/-146%, respectively. The stimulations of D, A, and B cells were abolished by propranolol.alpha-Adrenergic agonism (10 ng/ml epinephrine) after beta-adrenergic blockade) moderately decreased somatostatin (-37+/-7%) secretion, moderately increased glucagon (91+/-19%), and markedly decreased insulin (-85+/-3%) release. Similar effects on D-, A-, and B-cell secretion were induced with 2 ng/ml epinephrine or 10 ng/ml norepinephrine after beta-adrenergic blockade. The alpha-adrenergic effects on the D and A cell were abolished by either phentolamine or by phenoxybenzamine. This study showed that there are indeed alpha-adrenergic receptors on A cells and that the secretion of glucagon, a "stress" hormone, was stimulated either by alpha- or beta-adrenergic receptor agonism. D-cell secretion, like that of the B cell, was inhibited by alpha-adrenergic agonism and was stimulated by beta-adrenergic agonism. However, beta-adrenergic-induced changes in D-cell secretion were smaller in magnitude than those of B-cell secretion.

Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to beta-adrenergic stimulation.

It is unclear what factors control the secretion of pulmonary surface active material from alveolar type II cells in vivo. Other workers have suggested that cholinergic stimuli, adrenergic stimuli, and prostaglandins may all stimulate secretion. We isolated type II cells from the lungs of rats by treatment with elastase, discontinuous density centrifugation, and adherence in primary culture. beta-Adrenergic agonists, but not cholinergic agonists, caused an increase in the release of [(14)C]disaturated phosphatidylcholine, the major component of surface-active material, from type II cells in culture. The beta-adrenergic effect was stereo-selective, (-)-isoproterenol being 50 times more potent than (+)-isoproterenol. Terbutaline, 10 muM, a noncatecholamine beta-2 adrenergic agonist, caused a release of 2.0+/-0.5 (mean+/-SD) times the basal release of [(14)C]disaturated phosphatidylcholine in 3 h; the concentration of terbutaline causing half maximal stimulation was 800 nM. The terbutaline effect was blocked by propranolol, a beta-adrenergic antagonist (calculated K(d) = 6 nM), but not by phentolamine, an alpha-adrenergic antagonist. Isobutylmethylxanthine, a phosphodiesterase inhibitor, and 8-Br cyclic AMP, but not 8-Br cyclic guanosine monophosphate, also stimulated release. We conclude that type II cells secrete disaturated phosphatidylcholine in response to treatment with adrenergic stimulation.

Differentiation markers in fetal epidermis: transglutaminase and transpeptidase.

Two members of the transpeptidase family of enzymes, transglutaminase and gamma glutamyl transpeptidase, were assayed histochemically and biochemically in developing rat epidermis from day 15 of gestation through postnatal day 5. Electron microscopic examination of serial skin biopsies enabled precise dating of fetal epidermis and periderm and correlation of ultrastructural details of the cells with marker enzyme activities. Transglutaminase activity appeared histochemically in surface epidermis and in hair follicle inner root sheath on day 18 and day 21 of gestation, respectively, concomitant with the onset of terminal keratinization in these tissues. Enzyme activity was biochemically detectable 2 days before the histochemical stain became positive. Transpeptidase was active in fetal epidermis prior to keratinization but was only detectable in basal cells thereafter. Subsequent to birth, enzyme activity rose geometrically in hair follicles undergoing initial differentiation, and was thereafter found in all anagen hairs. Transglutaminase is active only in cells approaching terminal keratinization, while transpeptidase is associated with early phases of epidermal proliferation and differentiation.

Response of rat hepatic fatty acid synthesis and activities of related enzymes to changes in level of dietary fat.

The rate of in vivo fatty acid synthesis as well as the levels of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme (ME), citrate cleavage enzyme (CCE), acetyl-CoA carboxylase (ACX) and fatty acid synthetase (FAS) activities, have been studied in the liver of rats fed a fat-free diet for 7 days, followed by diets containing different amounts of soybean oil (0 to 24.79 kcal%) for 7 days. The dietary fat depressed activities of G6PD, 6PGD, ME, CCE, and FAS significantly at 1.24 or 2.48 kcal%. On the other hand, AC activity and the rate of fatty acid synthesis were decreased when the level of dietary fat was 12.39 kcal% or greater. These findings, as well as the pattern of decrement of enzyme activities and of lipogenesis, suggest a close correlation of fat feeding to ACX activity and fatty acid synthesis. The results also suggest that changes of G6PD, 6PGD, ME, CCE, and FAS activities may be largely independent of those modifications which occur in the substrate flux, concomitantly with the decrease of lipogenesis caused by the inclusion of fat in the diet.

The effects of alpha adrenergic agents on human platelet aggregation.

The effects of alpha adrenergic agonists and antagonists on human in vitro platelet aggregation were studied to characterize further the platelet alpha adrenergic receptor. Aggregation induced by ADP and U46619; a stable prostaglandin endoperoxide analog, was potentiated by alpha adrenergic agonists, an effect which was completely blocked by the alpha adrenergic antagonist phentolamine (1 X 10(-6) M) but not by prazosin (1 X 10(-6) M). The order of potency for the alpha adrenergic agonists in potentiating ADP-induced aggregation was clonidine greater than or equal to epinephrine greater than alpha-methylnorepinephrine greater than norepinephrine greater than phenylephrine greater than methoxamine. Epinephrine-induced platelet aggregation was blocked by phentolamine, yohimbine, dihydroergotamine, clonidine and lofexidine but not by phenoxybenzamine (1 X 10(-5) M). These findings suggest that: 1)clonidine and lofexidine are partial agonists and 2) that the alpha adrenergic receptor of the platelet is different from the classical postsynaptic alpha adrenergic receptor and more closely resembles presynaptic alpha adrenergic receptors.

Picrotoxin- and bicuculline-sensitive inhibition of cardiac vagal reflexes.

Transmission in the cardiac vagal reflex pathway can be inhibited by stimulation of the hypothalamic defense region or somatic afferent nerves. A pharmacological analysis of inhibitory modulation of reflex vagal bradycardia was undertaken in the present study. Picrotoxin (0.5--1.5 mg/kg i.v.) or bicuculline (0.5--1.5 mg/kg i.v.) produced a dose-related blockade of inhibition of reflex vagal bradycardia elicited by stimulation of the lateral hypothalamus or branches of the brachial plexus in spinal (C1 or C8 transected) cats. In contrast, strychnine and pentylenetetrazol failed to change the heart rate responses produced by stimulation of the hypothalamus or brachial plexus afferents. Picrotoxin and bicuculline also blocked inhibition of reflex vagal bradycardia produced by stimulation of the inferior olive in decerebrate spinal cats. This observation supports the contention that these agents act in the brain stem to block inhibitory modulation of reflex vagal bradycardia. In addition, picrotoxin and bicuculline lowered basal heart rate in spinal cats but not in decerebrate spinal cats. This observation suggests that tonic suprabulbar inhibition of reflex vagal bradycardia also is sensitive to blockade by picrotoxin and bicuculline.

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.

[Clinical Investigations about the behaviour of pulmonary respiration and cardiovascular circulation after the application of Rohypnol for basis-sedation for microsurgical operations in otolaryngology in local anesthesia (author's transl)].

The influence of analog-sedation on pulmonary respiration and cardiovascular circulation caused by the action of pentazocine/flunitrazepam respectively in microsurgical operations under local anesthesia in otolaryngology is investigated in two collectives of patients. The results are presented and the hazards, especially the hypoxemia and its avoidance are discussed.

Physiological mechanisms of flooding (implosion) therapy.

Desensitization of psychological and physiological complex structures may be the most important element of flooding treatment. The implosive sessions are assumed to represent a supramaximal stimulation of pathologically excited and inert complex structures resulting in protective inhibition, irradiation of excitation, reduction of the excitation and inertness, and a decrease of the overshooting autonomic reactivity of the complex structures, leading to reduction of anxiety, aggression, and other pathologically increased feelings. Advantages such as stronger and improved flooding can be achieved by a flooding in hypnosis. The therapeutic indications go beyond the usual treatment of phobic states. In order to establish the psychological and physiological mechanisms in implosion there is a need for psychophysiological investigations. However, much is unknown about mechanisms. Controlled comparisons with other treatments give limited answers. Perhaps an international case history bank might establish which clinical conditions might benefit by technical modifications of flooding.

[Effect of a chronic alpha adrenergic receptor blockade on basal secretion of renin in essential hypertension].

To investigate the effect of chronic alpha-adrenergic receptor blockade on renin release, plasma renin activity (PRA) was determined overnight at short intervals in 9 patients with essential hypertension before and after 7 days medication with phenoxybenzamine (20 mg orally/day). Inhibition of basal (supine) renin secretion in response to alpha-adrenergic receptor blockade was more apparent in patients with elevated PRA (n = 3) than in those with normal PRA. On the other hand, patients with low PRA (n = 2) even showed an increase in renin release. In addition, night-day variations with secretory episodes in PRA were blunted during drug administration. It is suggested that alpha-adrenergic receptor blockade inhibits renin secretion distal to its blockade of specific adrenergic receptors. However, the increase in renin release during phenoxybenzamine observed in patients with low PRA indicates that the responsiveness of renin secretion to stimulatory effects (most probably induced by the lowered blood pressure in our patients) remained intact during alpha-adrenergic receptor blockade.

Some biochemical parameters for qualification of bull semen.

Since the macroscopical and microscopical examination of bull semen does not in all cases appear to give conclusive indications concerning the fertilizing capacity, an investigation of some biochemical parameters was undertaken. It was shown that the following biochemical examination could furnish some more information about the quality of the semen: (1) Determination of the phenylalanine- alpha-ketoglutarate transminase activity both in the seminal plasma and in the whole semen. A high transminase activity in the plasma points to leakage out of the spermatozoa, thus indicating an affection of the spermatozoa. In that case larger amounts of basic amino acids and of leucine were often found in the seminal plasma than would normally be observed. (2) Gas chromatographic examination of the steroids occurring both in the seminal plasma and in the spermatozoa. In a number of infertile bulls small peaks of progesterone were found together with those of other, unidentified compounds. (3) Investigation of the carbohydrate metabolism in the semen. In a number of fertile bulls the following deviations were found: a. the presence of quite a lot of fructose in the spermatozoa; b. the presence of glucuronic acid and/of other foreign compounds in the spermatozoa or in the plasma; c. an elevated pH in the seminal plasma some 2 to 3 hours after ejaculation; d. a low phenylalanine- alpha-ketoglutarate transaminase activity in the whole semen (the spermatozoa had been disrupted by freezing). (4) Investigation of the presence of reducing aldehydes (glycoladehyde and glyceraldehyde) inside the spermatozoa. The determinations mentioned under 1, 3c and 3d can easily be carried out in the laboratories of each District Animal Health Service and of the A.I. stations; the same is true of the occurrence of amino acids in the seminal plasma and of fructose and aldehydes inside the spermatozoa if facilities are available for electrophoresis and chromatography.

[Acute pharmacotoxic psychoses in patients with chronic cerebral disorders].

269 patients suffering from progredient, chronic either primary or secundary cerebral diseases (Parkinson's disease, cerebral vascular diseases, cerebral atrophic dystrophy, Huntington's chorea, muliple sclerosis have been studied in the last two years. 44 of these patients developed pharmaco-toxic psychoses during drug treatment (low and medium dosis). The psycho-pathological rating resulted in an acute organic brain syndrome with predominance of confusion, sometimes progressing to delirium. EEG was changed during the psychotic stage. These changes cannot be decided from organic psychoses, which are not related to drugs. Patients with Parkinson's disease showed a relatively high incidence to psychoses during drug treatment (51.47%). In patients without Parkinson's disease, but on treatment with antidepressants, neuroleptics, diuretics and digitalis, pharmacotoxic psychoses only could be observed in 4.4% of the patients. However, the same group of patients showed an acute organic brain syndrome in 12.43%, when not on treatment. Combined treatment with L-DOPA plus peripherally acting decarboxylase inhibitors resulted in a high incidence to psychoses in idiopathic Parkinsonism but the same dosis produced this side effect only in a few patients with cerebral atrophic dystrophy. The ratio was 5:1 between the former group and the later one. That means, that L-DOPA is a much more psychotoxic substance in Parkinsonism when compared to other cerebral diseases. These pharmacotoxic psychoses could be correlated with the progredience of the disease. These pharmacotoxic psychoses are not only dependent from age and duration of treatment. Evidence exist, that there might be a correlation between the incidence for pharmacotoxic psychoses and the lack of surviving dopaminergic neurons in the nigro-striatal areas. Treatment with very low doses of neuroleptics suppresses pharmacotoxic psychoses but allow a further anti-Parkinson therapy which is of vital necessity.

[The influence of hypnotics on the development of morphological and biochemical wound reaction (author's transl)].

The influence of hypnotics (barbital and carbromal) on the development of the early wound reactions in mechanically injured skin of guinea pigs was investigated: 1) The cellular reactions in incised wounds were retarded after moderate intoxication by hypnotics (300 mg/kg barbital; 3 g/kg carbromal). The histomorphological changes in wounds were inhibited by carbromal twice as much as by barbital. The hitherto published investigations had not shown any retardation of the early cellular inflammation by weakening influences such as loss of blood or alcohol without symptoms of shock (Berg et al., 1977) or local disturbances by acids and bases (Kampmann et al., 1978). 2). A moderate delay of the activity of structure bound enzymes was found in barbital intoxication, a stronger restriction under the influence of carbromal. 3) After barbital intoxication significant elevation of histamine or serotonin in wounds was not seen. Under the influence of carbromal there was also no increase of histamine but an increase of serotonin. Thus, although the cellular reactions seem to be the most reliable indicator among the methods for the determinations of wound age under devitalizing influences, their value is reduced in cases of intoxications by hypnotics needing treatment. Possible pathophysiological connections of the alterations of morphological and biochemical wound reaction with shock are discussed.

[The effect of organic phosphoric esters on the experimental peptic ulcer (author's transl)].

The authors have investigated some organic phosphoric esters such as dimethoate, malatione, and trichlorphone in order to see whether they influence the genesis of the reserpine ulcer induced in rats or whether they act on the existing ulcer. Another objective of the investigation was to determine whether the aggravating effect of these compounds on the experimental gastric ulcer can be mitigated or prevented with isopropamide and gastrixon, two compounds known to be used in the therapy of ulcers. It was found that 1. dimethoate and trichlorphone considerably aggravate the genesis of the experimental peptic ulcer in rats, and that also malathione exercises this exacerbating effect still to some extent, 2. that the already existing experimental ulcer is aggravated by all three organic phosphoric esters and that 3. the severe experimental damage to the stomach can be reduced or prevented with Isopropamide and even more effectively with Gastrixon. As to the interpretation of the phenomena observed the authors take the view that--provided the effects mentioned apply also to man--with this a new risk was detected in the field of the chemical plant protection which may well gain importance for the health of a population.

Pharmacokinetics of femoxetine in man.

The pharmacokinetics of a structurally new 5HT-uptake inhibitor, femoxetine (FG 4963), with antidepressant properties have been investigated in man using a radioactive as well as a non-labelled substance. A two compartment open model gives a good description of the data, both after oral and intravenous administration. The substance was almost completely absorbed after an oral dose, but only 5-10% reached the systemic circulation due to extensive first pass metabolism. The metabolites had distribution and excretion rates similar to the parent compound. Only a small part (less than 2%) was excreted as femoxetine in the urine. The urinary excretion of the parent compound varied more than a 100-fold depending on the pH of the urine. The urine pH, however, did not influence the plasma concentration of femoxetine. Most of the substance (up to 80%) was eliminated by urinary excretion of metabolites, and only a small part of the radioactive dose was excreted in the faeces (up to 11%). The pharmacokinetic parameters were not found to be dose dependent in the range investigated, but it was not possible to decide whether the bioavailability was dependent on the dose. The variation between subjects was rather large, giving only a limited possibility for prediction of the plasma concentration from one subject to another.

Clinical significance of hyperparathyroidism in familial multiple endocrine adenomatosis type I (MEA I).

In order to investigate the suggestion that hyperparathyroidism in patients with familial MEA I has a mild and nonprogressive clinical course, we have compared clinical, biochemical, roentgenologic and histologic features of 29 patients with hyperparathyrodism originating from six families with the MEA I syndrome with those of 28 unselected patients with isolated nonfamilial hyperparathyroidism. The patients from the families with MEA I were significantly younger, had lower serum calcium and inorganic phosphate concentrations and a lower incidence of elevated alkaline phosphatase levels. Furthermore, they had multiple enlarged parathyroid glands and recurrence of the disease significantly more often. There was, however, no significant difference in the incidence of renal impairment, urolithiasis, subperiosteal resorption or large bone cysts on roentgenograms, histologic changes in bone biopsy specimens or mortality due to hyperparathyroidism. Therefore, the suggestion that this type of hyperparathyroidism has a milder clinical course is not confirmed in the present study.

Drug uptake into everted intestinal sacs. II. Inhibition of secretion by hypertonicity.

Mucosal hypertonicity, metabolic inhibitors, or absence of glucose and oxygen enhance mucosal-to-serosal influx of the cationic drug, pralidoxime (PAM), into sacs of everted rat jejunum in vitro. Conversely, efflux of PAM, which is twice the influx rate, is inhibited by mucosal hypertonicity or cyanide and iodoacetate. When sacs containing PAM, 0.87 mM, and glucose, 10 mM, were placed in identical drug- and sugar-containing mediums, the inside (serosal) concentration of PAM fell by over half in 120 min, whereas that of glucose more than doubled. Mucosal hypertonicity depressed PAM efflux and glucose influx regardless of serosal osmolarity. Although azide and mucosal hypertonicity each depressed glucose uptake and oxygen consumption while accelerating net PAM influx, azide more effectively depressed glucose and oxygen uptake, whereas hypertonicity caused greater acceleration of PAM uptake. Hypertonicity did not affect PAM binding to intestinal tissue. Varying mucosal pH did not change PAM or glucose uptake. Thus, mucosal hypertonicity apparently enhances net mucosal-to-serosal transfer of PAM by blocking its active secretion from serosa to mucosa.

[Influence of centrophenoxin administered for one year in high dose on maximal oxygen consumption in aged persons (author's transl)].

The influence of centrophenoxin (meclofenoxate) administration in a high daily dosage of 3 grams has been investigated in 10 persons with a mean age of 64 throughout 12 months. The mean age of the control group was 59. The function of bone marrow and a number of indicators of renal and hepatic function has shown no harmful changes after this long term treatment with an extremely high daily dosage. To investigate a possible influence on aging we chose the oral glucose tolerance test, a test battery for pulmonary function and the maximum oxygen consumption capacity. A highly significant (2P smaller than 0,335) influence of the drug for increasing the maximum oxygen input has been found. The hypothesis is presented, that this effect is due to an increase in cardiac functional capacity. Furthermore a significant decrease in fasting glucose levels has been found, while the glucose concentration one and two hours after administration of 100 grams of oral glucose have shown no significant changes. Body weight revealed a small but significant decrease. Side effects: we found a mild gastric pain in 4 patients that disappeared after 20 minutes. 5 Patients complained of a very small increase in jitteriness.

[Effect of dobutamine on intra-pulmonary shunt].

A study of the development of intra-pulmonary shunts was carried out in ten subjects following an intra-venous administration of a 7.5 microgram/kg/min dose of dobutamine by electric syringe. All the subjects were on a respirator (FiO2=0,6 most often) and frequently with P.E.E.P. The shunts were determined at the FiO2 by which the subject was being treated. The cardiac flow was measured by thermodilution. In 9 cases out of 10 the intra-pulmonary shunt is sharply increased already in the first half hour by dobutamine. The shunt values are in the order of: -- 17.94p. 100 +/- 7.19 before the drug -- 26.50 p. 100 +/- 12.85 half an hour after the infusion beginning. The shunt increase is thus 47p. 100 of the average original value after half an hour, which is significant (p less than 0.01). The shunt is then stable at the attained value (hourly average : 26.05 +/- 12.25). These observations are discussed in relation to the obtained effects on the cardiac outout, to the PaO2, and to the quantity of transported arterial oxygen. It appears in this series that, in spite of the shunt increase, there is no risk of cellular oxygen deficiency.

[Value of and indications for noxythiolin in generalized peritonitis].

The favourable experience of M. BROWN with noxythiolin in fecal peritonitis has prompted us, over the last seven years, to use this product in all forms of generalized peritonitis. This study involved 187 cases of generalized peritonitis : 102 males and 85 females, whose mean age was 53 years. Noxythiolin was reserved for severe cases (after having deliberately decided not to perform a comparative control series), the severity being related to the advanced age of the patients, the delay in intervention, and preexisting disorders. It was used in the form of a peroperative lavage of the peritoneal cavity and a postoperative instillation. The overall survival (71 p. 100) and that observed in particular in peritonitis of colonic origin (58 p. 100), seem to be arguments in favour of the beneficial effect of the product. Noxythiolin contributes to the sterilization of the peritoneal cavity. By virtue of its efficacy, the simplicity of its use, and its innocuousness, it merits a place in the therapeutic arsenal for treatment of generalized peritonitis, in combination with systemic treatments.

Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuliger on potato processing wastewater.

The yeasts Candida utilis and Saccharomycopsis fibuliger were propagated as a source of single-cell protein in a continuous, mixed, aerobic, single-stage cultivation on blancher water generated during potato processing. A series of steady-state experiments based on a two-level factorial design, half-replicate modified with an intermediate experiment, was performed to determine the effect of pH, 3.8 to 4.8; dissolved oxygen, 42 to 80% saturation; dilution rate, 0.17 to 0.31 h(-1); and temperature, 27 to 32 degrees C on the amount of carbon consumed, the rate of carbon consumption (R(c)), the amount of reducing sugar consumed, the rate of sugar consumption (R(g)), the amount of protein produced, the rate of protein production (R(p)), the yield from carbon, and the yield from reducing sugar. The results were analyzed by stepwise multiple regression and Fisher's least significant difference test. Analyses showed that high dilution rates resulted in increased R(c), R(g), and R(p) and indicated that a rate of 0.31 h(-1) was below the critical dilution rate. A temperature of 32 degrees C increased the amount of carbon consumed by 34%. A pH of 4.3 to 4.8 increased the amount of protein produced. The yield from carbon was constant, and the relatively high yield from reducing sugar indicated that other substrates were consumed. Dissolved oxygen was in excess at 42% saturation and above. Since C. utilis predominated the mixed cultures and amylase production appeared to be limited, a single-stage fermentation lacked efficiency. The experimental design allowed preliminary optimization of major environmental variables with relatively few experiments and provided a basis for future kinetic studies.

[Effects of beta blockaders on ventilatory function in chronic bronchitis].

This study was carried out to determine whether beta blockers could be prescribed for patients with coronary insufficiency and chronic bronchitis. The effects of intravenous infusions (30 mn) of propranolol (30 micrograms/kg), practolol 90 micrograms/kg), atenolol (90 micrograms/kg) and acebutolol (150 micrograms/kg) on vital capacity and expiratory flow rates were investigated in chronic bronchitics. Propranolol (n = 51) moderately reduced the vital capacity and FEV1, by an average 9% and a maximum of 20%. The three other infused agents given to groups of 10 patients did not change the ventilatory function. When the same patients were investigated by cross over with propranol bronchoconstriction was observed. This effect was seen in all stages of chronic bronchitis but was much less severe than in a group of 50 asthmatic patients (-23%). The respiratory tolerance of the cardioselective beta blockers seems to be better but there is considerable individual variation and the diagnosis between asthma and chronic bronchitis may itself be very difficult.

[Differentiation of muscle fibers of various types during postnatal development of the ventral serrate muscle in a rat].

In the denticulate ventral muscle of Wistar rats at the age of 1 day--2 months activity of NAD-N-dehydrogenase, succynic dehydrogenase and cytochrome oxidase has been determined in transversal cryostat sections. Quantitative estimation of the enzymes activity has been carried on by the plag-method. With age, general tendency to increasing activity of the enzymes mentioned is noted, but the dynamics of the increase is peculiar for every enzyme. Analysing the histograms on muscular fibre distribution according to their optical density, it is possible to estimate the dynamics. Simultaneously, the width of variational series, central statistical moments, indices of asymmetry and excess are also estimated. The whole course of the muscular fibre development, in accordance to the range and moments of distribution, can be devided into four main stages: stable, initial stage, slow increase of events, rapid increase of events and stabilization of the process. The stages mentioned pass gradually one into another making it possible to mark transitional stages (5--11, 15--19, 34--60 days). Using standard indices, it is possible to characterize more strictly the processes occurring in the course of muscular fibre differentiation. Lack of parallelism in the dynamics of asimilarity and excess can be treated as variety in differentiation of muscular fibres with middle and large optic density, and parallelism in dynamics--as their simultaneous differentiation. By comparing the curves it can be concluded that up to the 12--14th days, variety in differentiation of muscular fibres occurs, while after the 14th day their differentiation is more regular and simultaneous. The method of cytophotometry with subsequent mathematical processing of the results helps to determine the stages of muscular fibres differentiation.

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.

Kinetics of degradation of cefazolin and cephalexin in aqueous solution.

The kinetics of degradation of cefazolin and cephalexin in aqueous solution were investigated at 60 degrees C and constant ionic strength over the entire pH range. The observed degradation rates were obtained by measuring the residual cephalosporin and were shown to follow pseudo-first-order-kinetics. They were influenced significantly by solvolytic and hydroxide ion catalysis. No primary salt effect was observed in the acid or basic pH region. Of the buffer systems employed in the kinetics studies only the phosphate buffer system showed a catalytic effect. The pH-rate profile for cefazolin showed a degradation minimum between pH 5.5 and 6.5. Cephalexin did not show a pH minimum in that region. The apparent energies of activation were determined for cefazolin and cephalexin at pH 5.5 and were calculated to be 24.3 Kcal/mole and 26.2 Kcal/mole, respectively. The agreement between the calculated theoretical pH-rate profiles and the experimental points for both compounds support the hypothesis presented concerning the reactions involved in their respective degradation pathways.

Toxicology of clobazam.

1 Extensive toxicological investigations of clobazam are summarized. 2 LD50 values after oral administration ranged from 100 mg/kg in the dog to 6000 mg/kg in the rat. 3 Long-term repeated dose studies have shown withdrawal effects in the dog and monkey (notably convulsions leading to death), similar to those known to occur with other benzodiazepines. 4 It is concluded that the overall safety and tolerability of clobazam have been demonstrated in the preclinical animal studies.

Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups.

The electrostatic treatment applied in the preceding paper in this issue [Matthew, J. B., Hanania, G.I.H., & Gurd, F.R.N. (1979) Biochemistry (preceding paper in this issue)] to the titration behavior of individual groups in human deoxyhemoglobin and oxyhemoglobin was applied to the computation of the alkaline Bohr effect at various values of ionic strength. The enhanced proton binding of deoxyhemoglobin in the pH range of 6--9 was accounted for at ionic strength 0.01 M by the effects of the unique charge distributions of ionizable groups in the two quaternary states. At ionic strength 0.10 M the effects of 2--4 bound anions had to be considered in addition in the deoxyhemoglobin charge configuration. At the higher ionic strength 10 groups per tetramer contributed to the Bohr effect, whereas 28 groups were contributory at the lower ionic strength. The ionic strength dependence of individual groups in the two tetrameric structures as well as in the alpha-chain monomer was explained in terms of the electrostatic treatment. This examination showed that the differences in electrostatic behavior of deoxy- and oxyhemoglobin follow from particular dissymmetries in their configurations with respect to charge and static solvent accessibility.

Effect of substrate properties on the activity of lysosomal cholesteryl ester hydrolase.

The effects of the substrate properties on the catalytic activity of lysosomal cholesteryl ester hydrolase from rat liver have been examined with three standard substrate types: vesicle, micelle and emulsion. The pH optimum of the enzyme coincided to 4.5--5.0 with the substrate types employed. The apparent Km values were 15.3, 14.3 and 7.3 microM for vesicle, micelle and emulsion substrates, respectively. In the systems used in this study reaction products, cholesterol and oleic acid, and the nonionic surfactant Tween 80 and Triton X-100 Had an inhibitory effect. The emulsifier phosphatidylcholine and the charged phospholipid phosphatidic acid stimulated the activity. The mixed micelle of sodium taurocholate and phosphatidylcholine was the most potent substrate vehicle. With dipalmitoyl phosphatidylcholine vesicles the enzyme showed maximal activity at the gel-liquid-crystalline transition temperature of the phospholipid. The possible physiological significance of the lysosomal cholesteryl ester hydrolase is discussed with special reference to the form of the substrate.

[Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation].

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.

Some observations on the calcium ion binding to the eggshell matrix.

The calcified matrix of the hen eggshell has been demineralized with the EDTA. Aliquots of this material are soluble in water and have been characterized by column chromatography and by chemical analyses. Of particular interest is the high hexosamine and uronic acid content, which confirms the protein-polysaccharide nature of this water-soluble material. The calcium ion binding to the eggshell matrix has been studied by the equilibrium dialysis technique at different pH values, with both free and blocked carboxylic groups. The material with the free carboxylic side chain groups binds more calcium ions with increasing pH value. When the carboxylic groups have been previously blocked with a water-soluble carbodiimide, the calcium ion binding rapidly decreases. The residual capacity to bind calcium ions in the material with the carboxylic functions modified is probably due to the sulfate ions. In agreement with previous observations on other calcified substrates, the calcium ion binding seems to depend on the presence of ionized carboxylic functions of the matrix.

Treatment plan for acute and chronic adrenergic poisoning crisis utilizing sympatholytic effects of the B1-B2 receptor site blocker propranolol (Inderal) in concert with diazepam and urine acidification.

In a large series of patients who have abused a variety of commercially available "amphetamine-like" agents as well as "street drugs" with CNS stimulant activity, the specific lytic effects of propranolol (Inderal) were utilized to reverse the dangerous hyperkinetic cardiovascular and frightening CNS phenomena noted in these non-comatose individuals. Presented here is a logical and clinically proven mode of therapy by which the authors have consistently, successfully, and safely managed such patients in "adrenergic crisis" by judicious titration of electrolytes, propranolol, and diazepam.

Pharmokinetics of 2'-deoxycoformycin in normal and L1210 leukemic mice.

Following intravenous administration, 2'-deoxycoformycin (0.25 mg/kg) was rapidly distributed to tissues of both normal mice and mice bearing L1210 leukemia cells and readily eliminated, primarily by urinary excretion. Elimination of 2'-deoxycoformycin from plasma was biphasic, and half-lives for the alpha- and beta-phases of 10 and 33 min for normal mice and 7 and 40 min for L1210-bearing animals. The volume of distribution at steady state was approximately 20 ml, suggesting that the drug was distributed in the total body water for both groups of mice. The kidney, liver, small intestine, spleen, thymus, and L1210 tumor had tissue/plasma ratios greater than or equal to 1 at 15 min after dosing. In both groups, greater than 90% of the dose of 2'-deoxycoformycin was recovered in the urine within 3 hr. As determined by bioautography of urine samples, no detectable metabolism occurred. The presence of the L1210 tumor caused changes in the tissue distribution of 2'-deoxycoformycin. At later time periods, tissues from tumor-bearing mice contained significantly higher levels of this drug when compared to normal mice. However, the tumor was without significant effect on blood levels or urinary excretion of 2'-deoxycoformycin.

Pharmacokinetis of alpha- and beta-isomers of racemic endosulfan following intravenous administration in rabbits.

The pharmacokinetics of the alpha- and beta-isomers of endosulfan in rabbits was investigated following intravenous injection. Endosulfan (2 mg/kg) was given as a mixture of the two isomers in the ratio 70:30. The plasma concentration-time data for alpha-endosulfan was best fitted to the three-compartment model with a terminal slope half-life of 235 +/- 168 (SD; N = 6) hr. The data for beta-endosulfan was adequately described by the two-compartment model, the corresponding half-life was 5.97 +/- 2.41 hr. The total distribution volumes during the terminal slopes were, however, similar for alpha- and beta-endosulfan (675 +/- 246 and 565 +/- 126 ml/kg, respectively). Consequently, the plasma clearance was considerably lower for alpha-endosulfan (2.70 +/- 1.3 ml/hr/kg) than for beta-endosulfan (70.1 +/- 18.6 ml/hr/kg). The alpha-isomer had a higher fraction unchanged in the urine (37% of dose) than did the beta-isomer (11%) and a slightly higher fraction unchanged in feces (2.7% and 0.4%, respectively) at 5 days. Thus, the two isomers of endosulfan showed pronounced differences in their pharmacokinetic profile. These dissimilarities may partly explain reported differences in toxicity between the alpha- and beta-isomer.

Yeast argininosuccinate synthetase. Purification; structural and kinetic properties.

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228,000 as determined by gel sieving. It is composed of identical subunits of Mr 49,000 as shown by gel electrophoresis. The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric. The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate; it is increased by argininosuccinate and arginine. Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on the basis. Yeast argininosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.

Clonal dominance of low-affinity antibodies in rabbit hyperimmune anti-streptococcal group A-variant polysaccharide antisera.

Intraveneous hyperimmunization of selectively bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. In these antisera, IgG with dissociation constants Kd = 10(-6) M constitutes 90% and IgG with Kp = 10(-9) M accounts for only 10% of the group A-variant polysaccharide-specific antibodies. The low affinity antibody fraction represents the dominant clonotypes. Preparative isoelectric focusing in granulated (Ultrodex G-75) gels was used to successfully purify single-band material belonging to dominant spectrotypes. Affinity studies with these antibody fractions with the highest reported degree of purity yielded Kd = 10(-6) M values, thus confirming that clonal dominance is exclusively associated with low-affinity antibodies. Since it is known from previous work (M. Cramer and D. G. Braun, Scand. J. Immunol. 1975. 4:63) and from the rabbit antisera used here that clonal dominance of this sort is long-lived, this work fails to support the argument of immune maturation. The data more logically relate to antibodies that emerge with different subspecificities -- recognized in the antigen as a function of time in immunization procedures -- rather than to an inherent property of the immune system.

The effect of oral thyrotropin-releasing hormone on thyroid function and the composition of breast milk in puerperal women.

To determine the effect of oral administration of thyrotropin-releasing hormone (TRH) on the thyroid function and on the composition of breast milk in the early puerperium, six lactating women were treated with a single dose of 40 mg of synthetic TRH and six women were treated with placebo. Serial serum samples taken before and between one and 25 hours after TRH administration were assayed with specific radioimmunoassays for thyrotropin (TSH), triiodothyronine (T3) and total thyroxine (T4). Milk samples were collected three times a day and their major fatty acids were determined by gas-liquid chromatography and were compared with those obtained from normal lactating women. A statistically significant TSH elevation was observed between one and six hours after TRH administration, with a peak value of 23.7 +/- 10.6 mU/liter at three hours. The T3 concentration rose between three and nine hours after TRH administration, with a peak of 6.3 +/- 1.2 nmole/liter at six hours. The T4 elevation was statistically significant between six and 12 hours after TRH administration. The fatty acid content of milk samples from women treated with TRH did not differ from the normal series. A single daily dose of oral TRH thus caused a temporary thyroid stimulation. It is doubtful whether this could lead to hyperthyroidism since the levels of thyroid hormones became normal within ten hours after TRH administration.

Aspirin-stimulated intestinal electrolyte transport in rabbit ileum in vitro.

Because aspirin and the heavy metal salts of related anions have effects on intestinal ion transport and on diarrheal states, we have studied the effect of aspirin (ASA) on the in vitro rabbit ileum in an attempt to understand its mechanism of action. Ten millimolar of aspirin increased the conductance of rabbit ileum by 10--50% but did not change the permselectivity of the shunt path as determined by measurement of diffusion potentials. In Cl-free and HCO3-free solutions, aspirin reduced both Na absorption and the short-circuit current (Isc), which suggests an effect on electrogenic Na transport. Such an effect was not unexpected, since aspirin interferes with ATP production. However, in Ringer solution, aspirin in concentrations as little as 1 mM in the serosal solution reduced the Isc as before but also stimulated Na and Cl absorption and reduced JRnet (? HCO3 secretion) to zero. Aspirin had no effect on Na transport in the absence of Cl and no effect on Cl transport in the absence of Na, which suggests that aspirin stimulated a coupled transport process. Although these effects of ASA resemble those of alpha-adrenergic agents, ASA's effect was not blocked by alpha-adrenergic blockers such as phentolamine or phenoxybenzamine. The exact mechanism of ASA-stimulated NaCl absorption remains to be determined.

Neutrophil function and host resistance.

The part played by the phagocytic cells against invading pathogens has been known since the work of Metchnikoff nearly a century ago. This review deals primarily with the role of the neutrophilic polymorphonuclear leukocyte in host defense against microbial infections. The overall function of these cells in protection from infection is dependent on a number of steps. First, an adequate number of functionally mature neutrophils have to be produced and released into the circulation by the bone marrow. Cells must circulate normally and be capable of adhering to capillary and venule walls overlying inflammatory sites. The next step involves the exit of phagocytes from the blood stream through the capillary wall and emigration into the tissues to establish contact with the invading pathogens. This process is accomplished by the locomotive characteristics of these cells and chemotaxis. Most organisms must then be phagocytized to be killed. Two discrete phases are involved in phagocytosis; the "recognition" and attachment phase followed by the ingestion phase. After phagocytosis a series of coordinated morphologic and biochemical events are set into motion which leads to eventual death and lysis of the ingested microbes. A variety of antimicrobial mechanisms are involved in this final step and indicate that these cells have an appreciable reserve capacity if one mechanism is impaired. Recent evidence which clarifies mechanisms involved in all these stages is discussed.

Simultaneous localization of LHRH and catecholamines in rat hypothalamus.

The PAP unlabelled antibody enzyme method of Sternberger was used for the histochemical demonstration of LHRH and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) in the hypothalamus of the adult male and pregnant female rat. The sections for light and electron microscopy were serially treated with normal goat serum, LHRH antiserum and/or TH antiserum, goat anti-rabbit IgG, PAP complex and 3,3'-diaminobenzidine (DAB) or 4-Cl-1-naphtol. LHRH-positive cell bodies were discernible in the medial preoptic area. The LHRH-positive terminals were densely localized in the organum vasculosum of the lamina terminalis and in the perivascular region of the median eminence (PVME). Dopamine (DA)-positive cell groups (TH-positive perikarya) were discernible in the arcuate nucleus, and its terminals were densely localized in the PVME. The simultaneous identification of LHRH and DA in the distinctive neuronal system of the median eminence was possible with the PAP double staining technique, in which LHRH is revealed as a brown precipitate with DAB, and TH is revealed as a blue reaction product with naphtol. The LHRH neuronal system did not contain TH and vice versa. The ultrastructural study revealed that LHRH was localized in large vesicles with a diameter of 100 nm within the axon terminals, while TH was localized in the endoplasmic reticulum, the neurotubules and small vesicles with a diameter of 50 nm within the DA neuron. The axo-axonic contact of LHRH and DA terminals was demonstrated in close proximity to portal vessels, suggesting the synaptic influence of DA on the release of LHRH into these vessels.

Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro.

Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 43 degrees C but was only slightly affected by variation of the pH between 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin E1 or colicin E3.

A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney.

Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.

pH and bicarbonate effects on mitochondrial anion accumulation. Proposed mechanism for changes in renal metabolite levels in acute acid-base disturbances.

Mitochondria from rabbit and dog renal cortex were incubated with 1 mM (14)C-weak acid anions in media containing low (10 mM) or high (40 mM) concentrations of bicarbonate and the steady-state accumulation of labeled anion in the matrix was measured. In the absence of an energy source, no concentration of (14)C-anion in the mitochondrial matrix space was present, but the anion concentration was significantly higher at low- than at high-bicarbonate concentration. Addition of an energy source, usually ascorbate plus tetramethyl-p-phenylenediamine, led to increases in matrix space anion levels and to accentuation of the difference in anion uptake between low- and high-bicarbonate media, so that two to four times as much anion was present at low- than at high-bicarbonate concentrations. The anions affected included substrates for which inner membrane carriers are present in mitochondria, such as citrate, alpha-ketoglutarate, malate, and glutamate, as well as substances which diffuse passively across the inner membrane such as acetate and formate. When a nonbicarbonate medium buffered with Hepes was used, pH change did not alter anion uptake although anion concentrations exceeding those in the medium still developed when an energy source was present. The difference in mitochondrial anion accumulation between low- and high-bicarbonate levels diminished with decreasing temperature or with increasing anion concentration in the medium. Estimation of intramitochondrial pH with [(14)C]5,5-dimethyl-oxazolidine-2,4-dione showed that the pH gradient across the inner mitochondrial membrane was significantly greater with 10 than with 40 mM bicarbonate in the medium.A hypothesis is described that relates this effect of pH and bicarbonate on mitochondrial anion accumulation to the very rapid changes in substrate levels in renal cortex, which develop when acute metabolic acidosis or alkalosis is produced in the intact animal. It is suggested that an abrupt fall in systemic pH and bicarbonate is associated with a shift in substrate in renal cortex out of the cytoplasm and into mitochondria, where some of the added substrate is metabolized. Reduction in the size of the cytoplasmic pool of substrate occurs with relatively little accompanying change in the size of the mitochondrial pool, thus causing a net reduction in the total tissue pool. This mechanism accounts for the reduction in tissue levels of many mitochondrial substrates observed acutely in metabolic acidosis. In metabolic alkalosis, reversal of these effects leads to expansion of the cytoplasmic pool, thereby resulting in the rise in tissue levels of substrates which occurs in this condition.

Rapid diagnosis of meningitis with use of selected clinical data and gas-liquid chromatographic determination of lactate concentration in cerebrospinal fluid.

The usefulness of determination of lactate concentration in cerebrospinal fluid (CSF) for differentiation between various types of meningitis was evaluated. Lactate concentration in the CSF was assayed by gas-liquid chromatography for 115 patients, 17 of whom had serous meningitis and 38 had bacterial meningitis. The mean lactate concentration in the CSF of patients with bacterial meningitis was significantly higher than in the CSF of patients with serous meningitis and in a control group. The mean concentration in patients with serous meningitis was significantly higher than in controls. The highest lactate level in serous meningitis overlapped with the lowest level in bacterial meningitis. Elevated lactate concentrations in CSF were found also in patients with noninfectious disorders of the central nervous system. Misleading results may therefore be obtained if the lactate concentration in CSF alone is used to distinguish between serous and bacterial meningitis. The study suggests, however, that measurements of lactate levels in CSF, when combined with clinical and conventional laboratory observations, can increase the reliability of rapid diagnosis of bacterial meningitis.

Long-term haloperidol-treatment of mice: a change in beta-adrenergic receptor responsiveness.

Mice administered haloperidol 3 mg/kg/day in their drinking water for 21 days were tested for their locomotor responsiveness to saline or acid vehicle, dl-, l- or d-propranolol, metoprolol, butoxamine or practolol. Haloperidol-treated animals administered saline or acid-vehicle were, in five of six experiments, more active than animals withdrawn from vehicle-treatment. Haloperidol- and vehicle-treated animals responded differently to the non-selective beta-adrenoreceptor antagonists (dl-propranolol and l-propranolol) and selective beta1-adrenoreceptor antagonists (practolol and metoprolol), but not to a selective beta2-adrenoreceptor antagonist (butoxamine). With dl-propranolol (4 mg/kg) the locomotor activity of haloperidol-treated animals was significantly (0.01 less than P less than 0.02) greater than that of the vehicle-treated animals. Similar effects in the same direction were seen with l-propranolol (1 mg/kg, 0.005 less than P less than 0.01), practolol (10 and 100 mg/kg, 0.025 less than P less than 0.05 and 0.01 less than P less than 0.025 respectively) and metoprolol 8 mg/kg, 0.005 less than P less than 0.01). The d-isomer of propranolol which is about 50 times less active as a beta-adrenoreceptor antagonist than the l-isomer, although having equal membrane stabilizing effects, did not differentially affect haloperidol- or vehicle-treated groups. The results suggest that there has been a change in beta 1-adrenoreceptor responsiveness in animals withdrawn from long-term haloperidol treatment.

Effects of anti-anginal agents on cyclical reductions of coronary blood flow.

The effects of coronary vasodilating agents and alpha- and beta-adrenergic blocking agents on cyclical reductions of blood flow in the partially constricted coronary artery of anesthetized dogs were examined. Intravenous injections of nitroglycerin (50 microgram/Kg), SG 75 (150 microgram/Kg), papaverine (1 mg/Kg), and nicotinic acid (10 mg/Kg) eliminated both cyclical reductions of flow and ST elevation (group 1). Nifedipine (10 microgram/Kg), verapamil (500 microgram/Kg), diltiazem (500 microgram/Kg), and propranolol (500 microgram/Kg) suppressed ST elevation, but they could not eliminate cyclical reductions of flow (group 2). Dipyridamole (1 mg/Kg) and phenotolamine (500 microgram/Kg) augmented both ST elevation and cyclical reductions of flow (group 3). The results indicate that ST elevation due to cyclical reductions of coronary blood flow was eliminated by spasmolytic actions of group 1 on coronary artery, was suppressed by negative chronotropic and/or inotropic actions of group 2, and was augmented by peripheral actions of group 3.

[Hemodynamic and humoral changes during administration of a sympathomimetic and a sympatholytic drug with special notes on the regulation of renin release (author's transl)].

Studies in normal volunteers documented the positive inotropic effects of Etilefrin-HCL, a direct sympathomimetic drug, with increases of systolic blood pressure, renal blood flow and glomerular filtration rate. Sodium and potassium excretion as well as serum potassium decreased. After an additional injection of Metoprolol, a beta 1-sympatholytic drug, blood pressure, renal blood flow and glomerular filtration rate normalized, whereas electrolyte excretion decreased further. Renin release was decreased during administration of Etilefrin as well as during combined Etilefrin and Metoprolol application. Reziprocal to changes of blood pressure, plasma norepinephrine concentration decreased during Etilefrin and increased during combined administration of Etilefrin and Metoprolol. The results lead to the following interpretation: Changes of blood pressure and renal hemodynamics are mediated by beta 1-adrenergic effects of Etilefrin, whereas the electrolyte excretion is influenced by beta 2-adrenergic effects. Renin release seems to be influenced by beta 1 as well as beta 2-adrenergic receptors.