Lung bacterial clearance in murine pneumococcal pneumonia. We studied the bactericidal capacity of the rat lung during the development of pneumococcal pneumonia. Pneumonia was produced in a lower lobe by the intrabronchial instillation of 10(4)Streptococcus pneumoniae cells in buffer. Lung bacterial counts progressively increased, reaching 10(7) per lung within 48 h, and the increase was associated with localized atelectasis and consolidation. Bacterial multiplication was inhibited with tetracycline at various intervals after infection, and the subsequent clearance of pneumococci was determined. Viable pneumococci were rapidly killed by lung defenses if bacterial multiplication was inhibited within 12 h of the onset of infection. No change occurred in the bacterial populationif tetracycline was delayed until 24 h after infection, indicating that pneumococcal killing by lung defenses had ceased. This effect could be reproduced with the addition of pneumococcal capsular polysaccharide to the inoculum, which produced a dose-related inhibition of pneumococcal clearance. The clearance of S. epidermidis was not impaired in the presence of pneumococcal pneumonia or by administration of exogenous capsular polysaccharide. These data indicate that pneumococcal pneumonia causes a marked impairment in lung antipneumococcal defenses within 24 h of the onset of infection. This acquired defect in antibacterial defenses may be due to the accumulation of pneumococcal capsular material in the lungs of infected animals.


Mild alkaline hydrolysis of lipopolysaccharide endotoxin enhances its mitogencity for murine B cells. Mild alkaline hydrolysis was found to enhance the mitogenicity of lipopolysaccharide endotoxin for murine B lymphocytes. Alkaline treated lipopolysaccharide also retained its property as a polyclonal activator. Whereas this treatment reduced the lethality of endotoxin for mice, its toxicity for lymphocytes cultured in the absence of fetal calf serum was increased. Lipid analysis indicated that there were no significant changes in the fatty acids of lipid A, but particle size was significantly reduced and the material was more homogeneous and soluble than untreated lipopolysaccharide. The relationship of these effect on the structure of lipopolysaccharide endotoxin to the mechanism of B-lymphocyte activation is discussed.


Effect of some antihistaminic drugs on the oestrous cycyle of rats. The effect of some antihistaminic drugs namely antazoline hydrochloride, diphenhy, dramine hydrochloride and mepyramine maleate has been investigated on the oestrous cycle in albino rats. On daily intraperitoneal administration, for 6 days, all the three drugs (10 mg/kg) significantly (P less than 0.001) prolonged the duration of oestrous cycle. The duration of subsequent cycle returned to near normal.


[Assay of aminoglycosides in serum using the urease method (author's transl)]. The urease method developed by Noone et al. for rapid bioassay of aminoglycoside antibiotics in serum is described in detail. The accuracy of the method was improved by using a BM 1-09 electrode assembly (Metrohm, CH-9100 Herisau, Switzerland) in conjunction with a digital pH-Meter (Metrohm E 500). The mean difference between spiked serum samples and measured concentrations was +0.37 +/-0.78 microgram/ml. The coefficient of determination between the urease and agar diffusion method was 0.94. Disadvantages of the method are a low sensitivity at concentrations below 1.5 microgram/ml and a large serum sample (1.5 ml). Advantages are its simplicity and rapidity.


Circulating prolactin levels. I. Normal females. A specific, homologous radioimmunoassay was used to quantitate circulating levels of prolactin in females in various stages of life. They were grouped in periods of newborns to peripubertal females, reproductive, gravid and puerperal, and postmenopausal females. The results reveal dynamic changes of prolactin throughout various stages of life and support the mounting evidence that prolactin's role in humans might mimic that of animals. The role of prolactin in the ovarian cycle is unclear and will have to be ascertained at the molecular level.


Follicular development during late human pregnancy. In contrast to the accepted view of ovarian quiescence during pregnancy, the ovaries of preparturient women are covered with a dense population of small superficial follicles. In this study we have measured the follicular size and status of the oocytes and cumulus oophorus in 298 follicles from the ovaries of 30 women. The samples were taken at cesarean section and were examined with Nomarski differential interference microscopy. No oocyte was recovered from 50% of the follicles; 79% of the recovered oocytes and 78% of their cumuli were degenerative. Degeneration was correlated with appearance of phagocytes in the follicular fluid. These findings suggest that the endocrine status of gestation does not prevent early follicular development, but induces premature atresia.


Successful separation of X and Y and spermatozoa in human and bull semen. Using particle free semen extender and applying a low temperature thermal convection laminar flow to bring the sperm cells in vertical orientation and allowing them to act through the center of buoyancy, a bimodal distribution was achieved. Laser scanner for observing sedimentation pattern and F-body technique for identification of male sperm were used. Specimens from five human donors and ten bulls were separated, checked for purity, and frozen in liquid nitrogen. The lighter fractions contained 59.4% male (P less than 0.01), the heavier fractions 63.5% female (P less than 0.01), and the nonprocessed semen 48.0% male cells. Over 10,000 cells were counted to arrive at this statistically significant result. Observed sex ratio closely matched the predicted purity figures. Lighter and heavier fractions were further purified using forced convection galvanic method. Seventy to eighty percent purity in both fractions was confirmed by F-body and small cell galvanic migration tests with human as well as with bull semen.


Autoradiographic studies of rabbit ova after fertilization with thymidine-H3 and -C14 labelled spermatozoa originating from different bucks. Rabbit spermatozoa were labelled in vivo injecting male rabbits with thymidine-H3 and thymidine-C14. Ova fertilized with H3-labelled spermatozoa were found to contain more silver grains when they had cleaved several times than before cleavage. Fertilized ova originating from rabbits that had been mated with both an H3- and C14-thymidine labelled buck carried both labels. An explanation for this phenomenon may be that additional spermatozoa in the perivitelline space of fertilized ova pass labelled DNA-bases to the cleaving blastomeres.


Absence of a prenidatory effect of luteinizing hormone releasing hormone (LHRH) in hamsters. Luteinizing hormone releasing hormone at high doses will terminate gestation in rats during early and midpregancy (ED50 approximately equal to 100 microgram/day) and rabbits during early pregnancy. Early pregnancy in hamsters, in contradistinction, seems refractory to this effect. Administration of LHRH up to massive doses (10 mg/day) over the first 3 or 7 days of pregnancy failed to affect the pregnancies in meaningful fashion. Further, a single injection (100 mg) on day 5 had no effect on pregnancy; this system has been employed for the assay of prostaglandins because hamsters are remarkably sensitive to PG's (PGF2alpha, ED50 approximately equal to 17 microgram, PGE2, ED50 approximately equal to 210 microgram). The absence of response of hamsters to LHRH cannot be interpreted at present.


The role of prostaglandins on the increase in motility of the rat-uterine horn containing a silk suture. The presence of a silk suture in one uterine horn of the rats leads to an increase in spontaneous motility when compared with the contralateral control horn. Indomethacin, an inhibitor of prostaglandin synthetase inhibits the motility suggesting a role of prostaglandins in this process.


Paper chromatographic estimation of fructose and myo-inositol in human seminal fluid: a method for evaluating seminal vesicle and prostatic function. A semiquantitative paper chromatographic method for the separation of myo-inositol and fructose in human seminal fluid was described. The intensity of the chromatographic zones characteristic of myo-inositol, which is a marker for human prostatic secretion and for fructose, which is useful in evaluating seminal vesicle function, were estimated after staining with an alkaline silver nitrate reagent. Characteristic patterns were found for patients with prostatic or vesicular dysfunction, and for patients with agenesis or blockage of the vas deferens. Paper chromatographic analysis of split ejaculate samples allowed evaluation of the sequence of ejaculation of the prostate and seminal vesicles.


Gonadal function following vasectomy in the rat. Adult rats were studied at four, eight, and 12 months following vasectomy and sham-operation. The weights of the seminal vesicles, ventral prostate, pituitary, and kidneys were not significantly affected by vasectomy. Testicular endocrine function in vasectotomized rat was transiently stimulated as witnessed by elevation in testicular venous testosterone and androstenedione after four months. There then occurred signs of decline in gametogenic function and atrophy of the testis after 12 months whereas hormonogenesis appeared to remain at normal levels. There was no alteration in the morphology of the epididymis at any of the time intervals of study after vasectomy.


Testosterone production and metabolism in laboratory-maintained male rhesus monkeys. Plasma production rates (PR), metabolic clearance rates (MCR) and plasma levels of testosterone were determined in 10 male, laboratory-maintained rhesus monkeys on two occasions out-of-season (April and August) and once in-season (October). Plasma testosterone levels in October were higher than those in April and August. The plasma PR was unchanged in August, but markedly increased in October, as compared to April. The MCR showed a parallel increase betueen August and October. Thus changes in testosterone production and metabolism can be observed between in- and out-of-season male rhesus monkeys, maintained under strict laboratory conditions. These changes must be taken into consideration when the male rhesus minkey is used as an experimental model for human reproductive endocrinology.


Pituitary and ovarian response to acute stimulation with LH-RH in normal and anovulatory women. The LH FSH estradiol and progesterone responses to acute stimulation with LH-RH were studied in 12 normal women with ovulatory cycles (4 in the initial follicular phase, 4 in the mid-follicular phase and 4 in the late follicular phase) and in two castrated women, two under hormonal contraception, two with ovarian amenorrhea, twelve with central amenorrhea of no detectable origin (6 with normal and 6 with low basal gonadotrophins), eleven anovulatory patients with pseudomenstruation, two with anorexia nervosa, and two with pituitary amenorrhea. Each woman received a rapid i.v. injection of 100 microgram synthetic LH-RH at 9:00 a.m. Serum levels of LH, FSH, estradiol and progesterone were determined by radioimmunoassay in samples collected before and 60, 120, 240 and 480 minutes after injection. The findings were : 1) A significant rise in estradiol and progesterone levels, in addition to LH and FSH elevation, in normal women; 2) A lack of ovarian steroid response in the castrated women and in ovarian amenorrheas, which suggests that the source of steroid response to stimulation is not extragonadal; 3) Significant differences in the responses of the four hormones to LH-RH in the women with central amenorrhea in comparison with the normal group with great variability of results; the steroid response in the presence of a positive LH response might correlate with the severity and/or prognosis of the disorder, a point deserving further study; 4) In anovulatory women with pseudomenstruation, LH responses for the most part normal, and particularly, progesterone responses.


Seminal fructose and acid phosphatase in vasectomised men. Seminal fructose and acid phosphatase levels have been determined in 30 vasectomised and 30 nonvasectomised healthy adults, both the groups being closely matched for age (32 to 45 years). The fructose and acid phosphatase values in the vasectomised men, 3 to 6 months after the operation, were 316 +/- 36 mg% and 2,098 +/- 112 K.A. units/ml, respectively, while in the control group they were 251 +/- 28 mg% and 1,932 +/- 92 K.A. units/ml. The differences are statistically significant (P less than 0.001) and the possible implications of the post-vasectomy rise in these androgen dependent seminal constituents are discussed.


The 'in vivo' effects of oxytocin and vasopressin on spontaneous contractility of the rat epididymis. The spontaneous contractility of the epididymis in the rat was recorded in vivo and the effects of the neurohypophyseal hormones were studied. Oxytocin (50 muU and 500 muU/100 g body weight) produced a progressive increase in tonus together with an increase in amplitude and frequency of the contractions. Vasopressin (100 muU and 1000 muU/100 g body weight) showed similar effects. No differences were apparent at the doses studied.


Interpretation of thermal perturbation spectra of proteins. The thermal perturbation difference spectrum of reduced lysozyme has a long wave length extremum at 304 nm at pH 6.15 and a very small extremum at 306 nm at pH 1.5. These results differ from those of Leach & Smith (1972), which showed an extremum at 293 nm, the same as for model tryptophyl compounds. Our result may arise from a conformational difference between the two sample temperatures. The interpretation of thermal perturbation spectra of proteins is discussed. Contributions from thermally induced concentration differences, buried chromophores, and chromophores in  crevices are considered in the interpretation of the thermal perturbation spectrum of bovine serum albumin. It is suggested that chromophores in pauci-aqueous crevices may appear buried toward thermal perturbation spectroscopy but accessible toward solvent perturbation and chemical reagents.


Stabilisation of enzyme structures by inhibitors. A nuclear magnetic resonance study of the effect of phosphate on the acid unfolding of ribonuclease A. The acid denaturation of bovine pancreatic ribonuclease A in the presence of 0.2M sodium dihydrogen phosphate has been studied by n.m.r. spectroscopy. Phenylalanine, tyrosine and methionine resonances serve as monitors of the unfolding process. It is shown that the inhibitor shifts the equilibrium towards the native structure at acid pH. Exchange broadening of the C-2 resonances of the active site histidines, 12 and 119, occurs in the presence of phosphate, suggesting an equilibrium between native and unfolded structures. Stabilisation of the partially unfolded protein is observed at pH 1.5, as evidenced by the lack of the histidine resonance due to random coil protein. A scheme of the equilibria relating the various states of the protein is proposed.


Levels of neurotransmitters in brain of vitamin B12 deficient rats. The levels of norepinephrine, dopamine and 5-hydroxytryptamine in brain homogenates of vitamin B12-deficient rats have been investigated. The norepinephrine levels were significantly decreased in the deficient animals compared to controls. The two major catabolic pathways of norepinephrine e.g. monoamine oxidase and catechol-O-methyl transferase did not show significant variations. Both acetyl cholinesterase and butiryl-cholinesterase markedly decreased in the plasma of the vitamin B12-deficient rats.


The buccal absorption of ascorbic acid and its passage through lipoid membrane. Ascorbic acid concentration was measured in centrifuged and uncentrifuged saliva from normal young adult men and women. The salivary ascorbic acid content of uncentrifuged saliva was significantly higher in women on account of the ascorbic acid contained in the cellular sediment. Ascorbic acid absorption into the buccal mucosa was measured from solutions of pH range 3.4-9.0 held in the mouth for periods of 1-9 minutes. For a constant mouth contact time, increase in pH of the loading solutions resulted in reduction of buccal absorption, and diminishing transfer into the buccal epithelium. Percentage absorption was greater in males than females throughout the pH range. At pH 5, 80% of the loading solution was absorbed after 5 minutes. Diffusion of ascorbic acid through the lipoid membrane was more rapid in males. Female buccal cells had significantly higher ascorbic acid concentrations than male cells. Absorption of laevo and dextro ascorbic acid in human beings can be considered as partitioning into, or passage through, a lipoid phase into the buccal cells. The rate of passage of ascorbic acid into the buccal lining cells under controlled conditions of pH and buccal contact time is pH and concentration dependent in human beings.


Physicochemical characteristics, morphology and morphogenesis of virions of the causative agent of Crimean hemorrhagic fever. Similar physicochemical characteristics were found with strains of Crimean hemorrhagic fever (CHF) and Congo viruses. The particle size, sedimentation coefficient, buoyant density, weight of the particles as well as morphology and morphogenesis of these viruses were similar to those of other members of the Bunyaviridae family. Data are presented on reproduction of CHF virus in cell cultures and on the inner structure of its virion.


Studies of binding C3-substitute rifamycins to human and bovine serum albumin. The interactions of a series of C3-substituted rifamycins with human and bovine serum albumins were studied in order to find possible correlations between the degree of binding and the structural features of the various molecules. The results obtained indicate some of the physicochemical properties and, therefore, of the structural requirements which appear to determine or influence the bonding mechanisms of this series of rifamycins. Two types of interaction were found to exist, ionic and hydrophobic types. The findings suggest that the inhibition by protein of the antibacterial activities of these antibiotics depends on the type of bonding mechanism rather than the degree of binding.


On RNA-polymerases of leukemia L 1210 origin and an enzymatic method to screen antitumor antibiotics. Four DNA-dependent RNA-polymerases were separated from the cell homogenate of moust leukemia L1210 cell by DEAE-cellulose column chromatography and tentatively designated as Peaks I, II, III and IV in the elution order. Peak II was inactivated by the addition of alpha-amanitin and effects of antibiotics and enzymes on the RNA-polymerase activity using Peaks, I, II and a mixture of Peaks I and II were examined. The RNA-polymerases were used to screen for enzyme inhibitors produced by microbes. This enzymatic method was successfully proved to select antitumor antibiotics.


Pig plasma benzylamine oxidase: some considerations on the mechanism of the reaction. The kinetics of benzaldehyde formation during the oxidation of benzylamine by pig plasma benzylamine oxidase have been studied at different pH values. It has been shown that the first step of the reaction catalyzed by this enzyme is the formation of a Schiff base between the amino group of the substrate and the aldehyde group of the enzyme. Present kinetic studies are consistent with this mechanism and demonstrate the existence of two steps which are alternatively rate-limiting one below pH 6.0, the other above this pH value. The rate-limiting step above pH 6.0 requires the participation of OH-. A mechanism of reaction has been proposed in which the release of products follows the sequence: hydrogen peroxide, aldehyde, ammonia.


Oxygen consumption and ventilation of dogs during passive and active exercise. A comparison was made between the influence of passive and active exercise on the energy metabolism (EM) and respiratory parameters (RP) of dogs: respiratory minute volume and ventilatory equivalent for O2. Passive exercise was performed in 24 dogs, anesthetized with pentobarbital sodium and morphine or ketamine hydrochloride and pentobarbital sodium, by moving the four limbs at constant frequencies of 25, 45, or 80 movements/min before and after curarization. Active exercise was induced in 35 animals by electric stimulation of the quadriceps and triceps muscles or of the distal ends of femoral and radial nerves at the same frequencies previously indicated. Values obtained for EM and RP during passive exercise were not significantly different from those obtained during resting conditions; active exercise caused a significant increase which was directly proportional to the frequencies of limb movement. Our results indicate that passive muscle movement induces no alteration in O2 consumption and the possible relfexes generated in these experimental conditions were not apparently important on ventilatory control. In active movements, over the range of the frequencies studied, there is a proportional increase in O2 consumption followed by a corresponding increase in ventilation.


Rate of pH changes in blood plasma in vitro and in vivo. The rate of pH decrease in dog blood after addition of CO2 (dissolved in saline) was measured in vitro with a combination pH electrode and fast-response amplifier. The pH change was found to have an apparent half time of 6.0 +/- 0.5 s at 38 degrees C, similar to that predicted by previous mathematical simulations on the basis of uncatalyzed CO2 hydration kinetics and the buffering characteristics of plasma. Measurements were also made in vivo in dogs by withdrawing blood rapidly from a carotid artery through a temperature-controlled chamber containing a pH electrode. When the flow was stopped, we measured the pH change which occurs in blood (after it leaves the lungs) as the slow dehydration of bicarbonate continues. The measured half time was 7.3 +/- 0.6 s, again agreeing well with predictions. Essentially the same half time was found when the direction of approach to chemical equilibrium was reversed by adding CO2 to the inspired air. The rate of the reactions could be increased markedly by hemolyzing the blood to release carbonic anhydrase into the plasma.


Urease of Klebsiella aerogenes: control of its synthesis by glutamine synthetase. Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate.


Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness. Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.


Characterization of an endonuclease associated with the drug resistance plasmid pKM101. An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.


Enzymatic reduction of mercurous ions in Escherichia coli bearing R factor. Mercurous ion (Hg(+))-dependent reduced nicotinamide adenine dinucleotide phosphate oxidation was demonstrated in an extract from cells of Escherichia coli W2252 that bear R factor.


Base specificity of polyamine binding to synthetic polynucleotides. The binding of polyamines and magnesium to synthetic polynucleotides has been studied by gel filtration on a Sephadex G-50 column. Among the single-stranded polynucleotides examined [poly(A), poly(C), and poly(U)], polyamines were found to bind to poly(C) and poly(U) preferentially, while the binding of Mg2+ was greatest with poly(A). Spermine bound to poly(U) was displaced completely by NH4+ but incompletely by Mg2+, while Mg2+ bound to poly(A) was displaced completely be spermine but incompletely by NH4+. The optimal pH for the binding of spermine to poly(U) was found to be about 7.9, while Mg2+ could bind to poly(A) over a broad pH range (7.1--8.7).


Morphology of lipid micelles containing lysolecithin. Mixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0--6.4 nm repeat. The disc were 10--50 nm in diameter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0 degrees C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysolecithin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.


Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. I. Affinity labeling of glyceraldehyde 3-phosphate dehydrogenase and myokinase with a reactive AMP analog. Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by  a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes were favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The posibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.


Survey for alpha-(1 leads to 3)-glucanase activity among yeasts. Activity of alpha-(1 leads to 3)-glucanase was found in species of Cryptococcus, Rhodotorula, and Endomyces. Observations on the expression and stability of this enzyme in Rhodotorula minuta var. texensis was presented.


Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes. The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used  for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms. 4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon). 5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.


Chemical modifications of ribonuclease U1. In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.


Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp. Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.


Purification and properties of phenylalanine ammonia-lyase in cut-injured sweet potato. L-Phenylalanine ammonia-lyase (PAL) activity was developed in response to cut injury in sweet potato root tissue. The enzyme was purified from tissue incubated for 1 day after slicing by ammonium sulfate fractionation, column chromatographies on L-phenylalanyl Sepharose 4B, phosphocellulose. Sephadex G-200 and Sepharose 6B and preparative polyacrylamide gel electrophoresis. The molecular weight and sedimentation coefficient were estimated to be 285,000 to 320,000 and 11.6 to 11.9 S, respectively. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel yielded a single stained protein band which corresponded to a subunit weight of 80,000. Thus, the enzyme seems to be composed of four subunits of the same size. Neither L-tyrosine nor D-phenylalanine served as a substrate. Two Km values for the PAL were observed above and below 30 micrometers at various temperatures and were lower than those for PALs of other plants. The slope of the Arrhenius plot had a discontinuity at 17 degrees C. The values of activation energy were calculated to be 15,000 cal and 19,000 cal above and below 17 degrees C, respectively. Similar discontinuities were also observed in the effect of temperature on the Km values and the Hill coefficients. Negative cooperativity was observed at 10 degrees C (n = 0.83), but was not marked above 20 degrees C (n = 0.94).


The behavior of 9-aminoacridine as an indicator of transmembrane pH difference in liposomes of natural bacterial phospholipids. The behavior of 9-aminoacridine as an indicator of pH differences artificially set across a membrane has been reexamined in liposomes prepared from bacterial phospholipids extracted from chromatophores of Rhodopseudomonas capsulata grown photoheterotrophically. The dye behaves as an ideal indicator for pH differences lower than about three units; at higher pH's the expected linear dependence of Q/(100-Q) vs. pH is no longer strictly observed. Similarly a linear dependence upon the volume of the liposomes added has been verified. The amine ceases to respond to pH changes when the pH of the external medium exceeds the value of 10, corresponsing to the pKa of 9-aminoacridine. The apparent volume of the inner phase of liposomes, as calculated from fluorescence quenching, but not the slope of dependence of fluorescence on pH, appears to be affected by several factors, including the ionic composition, the osmolarity of the external medium, and the microscopic structure of the liposomes. Millimolar concentrations of earth-alkaline cations diminish the apparent internal volume of liposomes, in agreement with the complexing effect of these ions on phospholipid bilayers. The osmotic response of the apparent inner volume has also been verified; this parameter decreases linearly with the reciprocal of the external osmolarity, as expected from the van't Hoff relation; an osmolarity exceeding 0.3 M is, however, necessary in order to observe this effect.


CDP-diglyceride:inositol transferase from rat liver. Purification and properties. CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.


Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase. Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the  same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.


Nature of the lectin-induced activation of plasma membrane Mg2+ATPase. The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.


Purification of human erythropoietin.

Purification of human erythropoietin. Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecylsulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, were obtained at the last step of fractionation. The nature of the difference between these two components is not yet understood.


Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli. A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.


Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia. Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.


Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver. An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.


Selective de novo synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia after in vivo administration of nerve growth factor. Tyrosine hydroxylase is synthesized de novo in rat superior cervical ganglia in organ culture. The differential rate of synthesis is not increased significantly by the addition of nerve growth factor to the culture. Prior administration of nerve growth factor in vivo, however, leads to an augmented synthesis of tyrosine hydroxylase in ganglia subsequently cultured in vitro. The differential rate of tyrosine hydroxylase synthesis was increased by a factor of between 3 and 4. Increases in the differential rate of synthesis were detected within 6 h; the rate reached a maximum 24 to 36 h after a single injection of nerve growth factor. Administration of actinomycin D or of nerve growth factor antibody in vivo prevented the nerve growth factor-induced increase in the differential rate of tyrosine hydroxylase synthesis in vitro. However, the increase in the synthetic rate of tyrosine hydroxylase was not prevented by the addition of actinomycin D to the culture.


Solubilization and partial purification of heme oxygenase from rat liver. Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.


Biochemical and cytochemical evidence for ATPase activity in basal bodies isolated from oviduct. Biochemical and cytochemical techniques were used to determine whether oviduct basal bodies have ATPase activity. All studies were carried out on basal bodies isolated and purified from the chicken oviduct. These preparations contained structurally intact basal bodies with basal feet, rootlet, and alar sheet accessory structures. Whereas the specific activity of the basal body ATPase in 2 mM Ca++ or 2 mM Mg++, 1 mM ATP, pH 8.0, averaged 0.04 mumol Pi/min per mg protein, higher concentrations of either cation inhibited the enzyme activity. Furthermore, the pH optimum for this reaction was pH 8.5. In comparison, the ATPase activity in cilia purified and measured under conditions identical to those for determining the basal body ATPase activity averaged 0.07 mumol Pi/min per mg protein. However, the activity increased at higher concentrations of divalent cation, and the pH optimum was pH 10.0. By cytochemical procedures for localizing ATPase activity, ATP-dependent reaction product in isolated basal bodies was found to be confined to: (a) the cross-striations of the rootlet; (b) the outer portion of the basal foot; (c) the alar sheets; and (d) the triplet microtubules. It is concluded that basal bodiesve an intrinsic ATPase activity that, by a variety of criteria, can be distinguished from the ATPase activity found in cilia.


The activity of the pneumococcal autolytic system and the fate of the bacterium during ingestion by rabbit polymorphonuclear leukocytes. The extent to which autolytic microbial enzymes are involved in the fate of microorganisms ingested by phagocytes has not been determined. It is known, however, that activation of degradative enzymes occurs during certain microbicidal events. We examined the possible role of the pneumococcal autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) in the loss of viability and degradation of pneumococci during phagocytosis by rabbit polymorphonuclear leukocytes. Three bacterial systems were compared: (a) wild type pneumococci with an active autolytic system; (b) wild type bacteria grown under conditions that block the endogenous autolytic activity and (c) a mutant strain defective in the major autolytic enzyme of this bacterium. No differences could be detected between the autolysis-positive and negative bacteria in the rate of killing and in the fate of macromolecular cell constituents during ingestion by rabbit peritoneal polymorphonuclear leukocytes.


Hypopotassemia following open heart surgery by cardio-pulmonary bypass. 1. In this study where potassium concentration of perfusate of heart-lung machine was made high and no potassium was given until two hours after by-pass, the fall of plasma potassium level due to heart-lung bypass occurred in two stages, namely one hour of complete bypass and two hours after the end of bypass. 2. Regarding to the decrease of these plasma potassium level, increase of potassium excretion into urine and the movement of potassium from extracellular fluid into cells are presumed to be causes. Their genesis was discussed. 3. To prevent hypopotassemia following bypass, it is recommended to add KCl in the perfusate of heart-lung machine and to give intravenous drip of KCl immediately after bypass.


Renal enzymes in kidney cells selected by D-valine medium. The ability of D-valine medium to inhibit fibroblasts in cultures derived from kidneys of various mammals has enabled the selective proliferation of epithlial cells in the absence of fibroblast overgrowth. Studies of these selected epithelial cells have demonstrated the presence of D-amino acid oxidase, carbonic anhydrase, high levels of alkaline phosphatase and the renal specific pattern of lactate dehydrogenase. The presence of these renal enzymes suggests that the selected epithelial cells are of renal tubular origin and indicates that these differentiated functions are retained in cultured cells.


Interaction of local anesthetics with the transport system of glucose in human erythrocytes. Local anesthetics inhibit the exhange transport of glucose in human erythroytes. All compounds tested showed a competitive inhibition except lidocaine and baycaine causing a non-competitive one. Moreover the transport system can bind two inhibitor molecules to one transport site as described for tetracaine and oxybuprocaine.


Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity. Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.


Analysis of medazepam, diazepam, and metabolites in plasma by gas-liquid chromatography with electrolytic conductivity detection. An improved electrolytic conductivity detector allowed the gas-liquid chromatographic analysis of medazepam, diazepam, and major metabolites in 2 ml plasma at concentrations of 20 microgram/l. The detector had a sensitivity limit of less than 1 ng (or 100 pg nitrogen) when operated in the nitrogen-selective mode and a nitrogen/carbon elemental selectivity ratio of greater than 100,000 compared to octadecane and cholesterol. Detector response when operated in various element-selective chemical modes was investigated, and its application to the analysis of the title compounds was compared to electron capture and flame ionization detection systems.


Use of an amino-silica column for the high-performance liquid chromatographic analysis of synthetic oligodeoxy-nucleotides. The use of an amino-silica column in the chromatographic analysis of synthetic oligodeoxyribonucleotides and their derivatives from different stages of oligonucleotide synthesis has been investigated. By eluting with 0.10 M potassium phosphate solution of pH 3.30, the nucleotide composition of oligonucleotides can be established within 15 min. In a linear gradient of phosphate buffer (0.10-0.75 M) at neutral pH, the separation of oligonucleotides by length and in an acidic medium pH 3.30-4.30) by composition is possible; the oligonucleotides may be in the free form or modified by the various protecting groups used in synthetic oligonucleotide chemistry. The analysis of some reaction mixtures from different stages of oligonucleotide synthesis and of a number of synthetic oligodeoxyribonucleotides and their derivatives has been performed.


Cimetidine, an H2-antihistamine, stimulates prolactin secretion in man. Intravenous administration of cimetidine, a histamine (H2) receptor antagonist, provoked an immediate 2 to 3-fold rise in serum prolactin concentrations in 8 normal men; serum thyrotropin, growth hormone, thyroxine, and triiodothyronine were not significantly affected. Diphenhydramine, an H1-antagonist, was without effect on any of the hormones measured. The results suggest that histamine may have an important function in the regulation of prolactin secretion in man.


Value of sputum culture in diagnosis of pneumococcal pneumonia. In our laboratory, culture of sputum was extremely useful in diagnosing the etiology of pneumococcal pneumonia. Of 31 consecutive patients with bacteremic pneumococcal pneumonia, 29 (94%) had Streptococcus pneumoniae cultured from sputum. Recovery of pneumococci in culture was enhanced by anaerobic incubation as well as by a plate bile test and an optochin disk on a primary blood agar plate.


Cytochemical reactions of normal and neoplastic lymphocytes. Rosetting and non-rosetting lymphocytes collected from normal individuals were stained for the presence of beta-glucuronidase, periodic-acid Schiff activity, gamma glutamyl transpeptidase, acid phosphatase, and alpha-naphthyl butyrate esterase. Lymphocytes which formed rosettes with sheep erythrocytes and non-rosette forming lymphocytes contained cytochemical reaction products for all five stains. Beta-glucuronidase (P less than 0-02) and acid phosphatase (P less than 0-01) were more frequently found in rosette forming lymphocytes. However, non-rosetting cells were more frequently periodic-acid Schiff positive (P less than 0-001). Gamma-glutamyl transpeptidase and alpha-naphthyl butyrate esterase were present equally in rosette and non-rosette forming lymphocytes. In addition, 33 non-Hodgkin's lymphomas were studied for cell surface markers and cytochemical reactions. In 17 of 19 B cell lymphomas, there was a paucity of lymphocytes containing beta-glucuronidase. However, in three of four T cell proliferations, there were numerous lymphoid cells positive for beta-glucuronidase. The periodic-acid Schiff and acid phosphatase reactions varied greatly within B, T, and null cell lymphomas and thus were of little diagnostic value in determining the cell of origin of these neoplastic lymphoid cells.


The effect of a new benzodiazepine on the polygraphically monitored sleep of normal volunteers. All-night sleep was polygraphically monitored from ten normal volunteers who took placebo and three dosage levels of a new benzodiazepine hypnotic, SCH 16134, in a double-blind, crossover design. All dosages of the drug decreased the time to fall asleep, and the two highest dosages also decreased interspersed wakefulness. REM sleep was suppressed, but slow-wave sleep was not affected in this experiment. The subjective quality of sleep was improved by the new hypnotic. One subject reported that he felt lethargic the day after the largest dose.


Multiclinic double-blind comparison of triazolam and flurazepam for seven nights in outpatients with insomnia. In this two-clinic seven-day double-blind study, 0.5 mg triazolam (Halcion) was compared to flurazepam (Dalmane) in the treatment of insomnia. Two clinical investigators completed 118 outpatients, 61 on triazolam and 57 on flurazepam. Five patients, four on triazolam and one on flurazepam, discontinued because of side effects; and three patients, one on triazolam and two on flurazepam, discontinued because of ineffectiveness of the medication. Analysis of pooled data for the 110 evaluable patients showed that 0.5 mg triazolam was significantly better than 30 mg flurazepam on the following parameters: (1) how much the medication helped the patients sleep, (2) onset of sleep, (3) duration of sleep, (4) evaluation of duration of sleep, and (5) feeling of restfulness in the morning. The trend for all other parameters favored triazolam treatment, but the values did not reach statistical significance. Side effects were similar in both groups, with drowsiness being reported most frequently. No change in efficacy indicating tolerance development during the seven days of drug administration was observed in either group.


Effect of a new benzodiazepine derivate, clobazam, in anxious patients with gastrointestinal disorders. Thirty-four anxious patients with gastrointestinal disorders were studied in order to evaluate the effectiveness of a new 1,5-benzodiazepine antianxiety agent (HR 376). The disorders were classified as organic or functional according to the presence or absence of radiologic signs of ulcer. Dietetic measures, gastric antacids, anticholinergic agents, and antianxiety treatment were applied for six weeks. Anxiolytic treatment consisted of 30 mg/day clobazam (HR 376) or 15 mg/day diazepam, given in a randomized, double-blind manner. Clinical follow-up was performed with the PEN Personality Inventory (PEN), Taylor Manifest Anxiety Scale (TMAS), Hamilton Anxiety Scale (HAS), and Wittenborn Psychiatric Rating Scales (WPRS). The score of the psychoticism dimension of the PEN inventory was significantly higher in organic than in functional patients. Significant differences occurred in the reduction of the rating scores of HAS and WPRS before/after treatment in the clobazam and diazepam groups. This would express a modification of state anxiety. The TMAS, which evaluates trait anxiety, disclosed statistically significant improvement in the clobazam group. This group showed an early reduction of the HAS and TMAS scores, which would suggest an early onset of action.


Effect of electrical stimulation of the autonomic nerves on the levels and synthesis of cyclic nucleotides in the rat salivary glands: relationship to enhanced growth. Electrical stimulation of either the parasympathetic or the sympathetic nerve supply to the parotid and submaxillary glands increases the intracellular level of cyclic GMP and the rate of DNA synthesis and cell division while only sympathetic stimulation raises cyclic AMP levels. The periods of electrical stimulation inducing hyperplasia also raise the cyclic GMP concentration but there is no similar correlation with changes in cyclic AMP levels. However, the extent of hyperplasia induced by parasympathetic and sympathetic stimulation is not directly related to the size of the increase in cyclic GMP concentration that these treatments produce. Changes in cyclic AMP levels are reflected in altered in vitro adenylate cyclase activity. This activity is raised after 2 min sympathetic stimulation and markedly decreased with 30 min sympathetic or parasympathetic stimulation. Guanylate cyclase activity shows no such changes with nerve stimulation.


Fetal and maternal plasma electrolytes, blood gases, and pH in dairy cows during late gestation. Sodium, potassium, and chloride were determined in fetal and maternal plasma of six cows between 208 and 269 days of gestation. Blood pH and partial pressures of oxygen and carbon dioxide also were measured in samples collected from the jugular vein of the cow and the caudal aorta and vena cava of the fetus. Concentration of chloride in fetal plasma was generally lower than the corresponding maternal value, and fetal potassium was greater than maternal. Potassium of fetal plasma increased towards term. Sodium concentrations were not significantly different between fetal and maternal plasms. Once fetal and maternal pH and blood gas tensions had stabilized after surgery, there was little change until shortly before delivery. In the last 24 h before calving maternal partial pressure of oxygen was high and fetal blood pH was low.


Biochemical and histocytochemical studies on response of ammonia-producing enzymes for nh4cl-induced acidosis. NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.


Bunyavirus isolations from mosquitoes in the western Canadian Arctic. Strains of California encephalitis virus (snowshoe hare subtype) were isolated from 8 of 475 pools comprising 23 747 unengorged female mosquitoes of five species collected at three of six locations throughout the Mackenzie Valley of the Northwest Territories, Canada, from latitudes 60 to 69 degrees N between 10 and 24 July 1976. Minimum field infection rates included 1:2734 for Aedes communis, 1:256 to 1:3662 for A. hexodontus and 1:911 to 1:1611 for A. punctor. Northway virus was also isolated from 1 of 3662 A. hexodontus mosquitoes collected at Inuvik (69 degrees N, 135 degrees W). Transmission of CE virus by A. communis infected by feeding on virus in defibrinated blood and incubation at 0, 13 and 23 degrees C for 13-20 days clearly demonstrates the importance of this species as a natural vector, and transmission of CE virus by Culiseta inornata after incubation at 0 and 13 degrees C following intrathoracic injection strengthens evidence of its role as a natural vector. Immunofluorescence was less reliable than imunoperoxidase for detection of CE viral antigen in mosquito salivary glands.


Regulation of cell-mediated cytotoxicity. III. Synergism of cortico-resistant with anti-thymocyte serum-resistant splenic T cells in the generation of cytotoxic lymphocytes in graft-vs-host reactions. Lethally irradiated F1 hybrid mice were given an i.v. injection of parental strain spleen cells. Six days later, their spleen cells were used as the effector cells to measure the in vitro cell-mediated cytotoxicity (CMC) of the parental cells. The treatment of the donors with hydrocortisone resulted in a marked decrease of the capacity of their spleen cells to produce a CMC reaction, whereas the treatment with antithymocyte serum (ATS) resulted in an almost complete loss of such activity. The mixing of spleen cells from hydrocortisone-treated parental donors with the spleen cells from ATS-treated parental donors before injection resulted in a synergistic amplification of the cytotoxic response. The anti-Thy-1 serum treatment of either spleen cell population abolished the synergism completely. These results indicate that cortico-resistant T cells act as precursors of cytotoxic lymphocytes and that ATS-resistant T cells produce an amplification of their reaction.


Differential susceptibility of Escherichia coli and Proteus mirabilis to mouse urine and to urea. The purpose of this investigation was to compare the antimicrobial properties of mouse urine and of urea against Escherichia coli and Proteus mirabilis. Nornal urine was found to inhibit the growth of E. coli and P. mirabilis, whereas urine from diuresing animals permitted multiplication of these bacteria. Addition of urea to urine from diuresing animals restored its bactericidal effect on P. mirabilis but not on E. coli. This bactericidal effect on P. mirabilis was dependent on the additive action of high content of urea and high pH and was prevented by the addition of urease inhibitor.


Alteration of structure and penetrability of the vitelline envelope after passage of eggs from coelom to oviduct in Xenopus laevis. The vitelline envelope (VE) that surrounds an egg released from the ovary into the coelom of Xenopus laevis differs markedly, in structure and penetrability, from the VE surrounding an oviposited egg. In a coelomic egg, the filaments that form the VE are arranged in distinct fascicles or bundles. The exterior surface of the VE is irregular in contour and is permeated by channels. In an oviposited egg, the filaments are evenly dispersed and lack a fasciculated arrangement; the exterior surface is smooth and no channels are present. The fascicular arrangement of fibrils in the coelomic VE is maintained only at neutral pH, and is not visibly altered by the cortical reaction. VEs from coelomic eggs retain their fasciculated morphology after isolation from the egg. In an in vitro test system, sperm penetrated VEs isolated from oviposited eggs, but failed to penetrate VEs isolated from coelomic eggs. The structural transformation of the VE from the coelomic type to the oviposited type occurs in the first 1-cm segment of the oviduct, prior to addition of jelly to the egg. Neither intact jelly, solubilized jelly, nor jelly extracts were capable of altering the structural organization of coelomic VEs, suggesting that the structural transformation of the VE is effected by some oviducal factor other than jelly.


Ribonucleotide reductase from herpes simplex virus (types 1 and 2) infected and uninfected KB cells: properties of the partially purified enzymes. Mammalian ribonucleotide reductase is a complex enzyme modified in its activity by a complex regulatory system involving adenosine triphosphate (ATP) and deoxyribonucleoside triphosphates. Infection of KB cells with herpes simplex virus (HSV) type 1 or 2 induces the formation of an altered ribonucleotide reductase. The properties of partially purified reductase from uninfected KB cells have been compared with the enzymes obtained from HSV-1 and HSV-2 infected KB cells. We found that the virus-induced enzymes are similar to the KB enzyme in some properties but differed significantly from the host enzyme in three respects: (1) virus induced reductase was not inhibited significantly by deoxythymidine triphosphate regardless of ATP concentration, (2) magnesium was not required for virus enzyme activity although 2 mM-Mg2+ did stimulate the reaction, and (3) magnesium concentration required for optimal activity was different for virus and host enzymes. These changes are evidence that the enzyme molecules present after infection by HSV-1 or HSV-2 differ from those present before infection.


MMPI changes in briefly hospitalized non-narcotic drug users. The Minnesota Multiphasic Personality Inventory (MMPI) was administered to 66 non-narcotic drug abusers as part of an intensive study of polydrug abuse. Patients were classified into four primary drug-of-abuse groups using either stimulants, barbiturates, other sedative-hypnotics, or a combination of these drugs. It was readministered to 42 of these patients after 2 weeks' hospitalization. At admission, the group's MMPI profile was consistent with psychosis. At discharge, most of the MMPI T-scores were sharply reduced, and the groups' profile was consistent with sociopathy. The admission MMPI profiles of the four primary drug-of-abuse groups did not differ. At discharge, the stimulant group's profile remained psychotic, while the profiles of the other three groups were not psychotic. Such results raise the possibility of a toxic psychotic effect of chronic non-narcotic drug abuse. The group of stimulant abusers appeared schizophrenic, while the other groups of non-narcotic drug abusers appeared sociopathic.


Inhibition of catecholamine release by alpha-adrenergic activation: interaction with Na, K-ATPase. In rat adrenal gland incubated in vitro, phenylephrine and naphazoline inhibited the release of catecholamines (CA) induced in a medium containing K+ (5.6mM). In K+-free medium or in the presence of ouabain (10(-3) M) phenylephrine and naphazoline had no effect on CA release. The alpha-adrenergic blockers, phentolamine and phenoxybenzamine, increased CA release in a K+-medium but had no effect on CA release from adrenal medulla in a K+-free or ouabain (10(-3) M) containing medium. PGE2 inhibited the release of CA both in the presence and in the absence of K+, as well as in the presence of ouabain (10(-3) M). Activation of alpha-adrenergic receptors in the adrenal medulla by phenylephrine and naphazoline resulted in increased Na, K-ATPase activity but no effect on Mg-ATPase. PGE2 acts through a different mechanism.


Suppression by GABAergic drugs of the locomotor stimulation induced by morphine, amphetamine, and apomorphine: evidence for both pre- and post-synaptic inhibition of catecholamine systems. Locomotor stimulation induced in mice by morphine and amphetamine was antagonized by pretreatment with gamma-butyrolactone (GBL) and amino-oxyacetic acid (AOAA) at doses which had little effect on saline treated animals. The effects of morphine and AOAA on the turnover of brain catecholamines (CA) were determined by measuring both the accumulation of dopa after inhibition of central aromatic L-amino acid decarboxylase and by measuring the depletion of noradrenaline (NA) after inhibition of tyrosine hydroxylase by alpha-methyltyrosine (alpha-MT). Morphine and AOAA were found to have opposite effects on CA turnover, i.e. morphine caused an increase and AOAA, a decrease. AOAA also antagonized the morphine-induced increase in CA turnover. These data might suggest that the well documented ability of GABAergic drugs to inhibit the firing of DA-containing neurons may be of importance in explaining the present findings. However, the locomotor stimulation induced by the directly-acting CA agonists, apomorphine and clonidine after pretreatment with reserpine and alpha-MT was also inhibited by the GABAergic drugs. It is therefore concluded that the suppressant effects of the GABAergic agents on hypermotility and not solely mediated by their effects on presynaptic CA mechanisms, but also by a postsynaptic inhibition at some point beyond the CA neurons.


Metabolic fate of cyanocobalamin taken up by Spirometra mansonoides spargana. Analysis of tissue from Spirometra mansonoides spargana has shown that cyanocobalamin (vitamin B12) is metabolized to adenosylcobalamin and hydroxocobalamin. No methylcobalamin was detected. When the tissues were examined for enzymes which are known to utilize coenzyme forms of vitamin B12, only methylmalonyl CoA mutase, which requires adenosylcobalamin was found. The enzyme, tetrahydropteroylglutamate methyltransferase, which requires methylcobalamin as a cofactor, was not detected. A sizable portion of the cyanocobalamin taken up was bound to ammonium sulfate-precipitable material, suggesting that the binding substance is a protein. Vitamin B12 taken up by spargana was found to be released in vivo with a biological half-life of about 7 weeks.


The pharmacology of benoxaprofen (2-[4-chlorophenyl]-alpha-methyl-5-benzoxazole acetic acid), LRCL 3794, a new compound with antiinflammatory activity apparently unrelated to inhibition of prostaglandin synthesis. Benoxaprofen is a potent and long-acting anti-inflammatory and antipyretic compound. Its anti-inflammatory activity has been demonstrated in carrageenan-induced oedema, in cellulose pellet granuloma and in both developing and established adjuvant arthritis tests in rats. Its antipyretic activity is greater than either aspirin or paracetamol in tests inducing pyrexia with yeast of 'E' pyrogen in rats and rabbits. Benoxaprofen has analgesic activity in tests where pain is accompanied by inflammation but not in other experimental models of pain. The weak prostaglandin synthetase inhibiting properties of this compound differentiate it from other acid anti-inflammatory compounds. The low ulcerogenic potential of benoxaprofen seen in animal models may be related to its relative inability to inhibit PG synthetase.


The in vitro metabolism of 2-nitroso-1-phenylpropane dimer by fortified 9000g supernatants from rabbit liver. 2-Nitroso-1-phenylpropane dimer, a postulated metabolic product of amphetamine, was metabolized by fortified 9000 g supernatants from rabbit liver, mainly by oxidation on the carbon beta to the nitrogen to form a "beta-hydroxylated nitroso dimer'. The corresponding nitro compound, ketone and alcohol were also products, the latter two but not the former being cofactor dependent. The nitroso compound was not metabolically reduced under the conditions used.


Application of orthogonal functions to the spectrophotometric determination of phenytoin and phenobarbitone in pharmaceutical preparations. Orthogonal polynomial functions have been used in the simultaneous determination of mixtures of phenytoin and phenobarbitone in pharmaceutical preparations. The mean recovery (9 determinations) and coefficeint of variation were 100-0 +/- 1-4% for phenobarbitone and 100-0 +/-1-3% for phenytoin. Neither the delta A method of Pannerdan & Fontein nor a delta A modification of Vierordt's method of two-component analysis gave results of the same accuracy and precision as the proposed method.


Autoxidation of N-hydroxyphenylalkylamines: the inhibitory effect of some anions on copper catalysed  autoxidation of N-hydroxyphentermine. The inhibitory effect of certain electrolytes and buffers on the copper catalysed autoxidation of N-hydroxyphentermine (2-hydroxylamino-2-methyl-1-phenylpropane) has been investigated. The presence of ions such as SO4(2-), Cl- or Br- markedly reduced the rate of oxidation. Phosphate and carbonate buffers had a similar effect with halides and phosphate buffers being the most inhibitory. The occurence of 2-methyl-2-nitro-1-phenylpropane and 2-methyl-1-phenylpropene-(1) as secondary oxidation products was also established.


Generated surface area measurement of disintegrating tablets. The surface areas generated by the disintegration and dissolution of six commercial brands of phenylbutazone tablets B.P. 100 mg have been measured using a Model TA Coulter Counter. The graphs of surface area generated against time were all of the same shape and always reached a maximum value. The initial surface area increase was due to tablet disintegration and deaggregation and followed first order kinetics. A good correlation between the slope of this initial increase and disintegration time was found. The maximum surface area generated correlated at the probability level of better than 99-9% with the dissolution rat measured as t60. The subsequent decrease in surface area, after this maximum, was considered to be due to phenylbutazone dissolution and was also first order rate controlled.


Hydrolysis and epimerization kinetics of hetacillin in aqueous solution. Methods were developed for quantitating epimerization to epihetacillin and hydrolysis to ampicillin in the alkaline degradation of hetacillin, and both rates in deuterium oxide at 35 degrees and in water at various temperatures were determined. In each case, plots of log k for the epimerization against pH or pD yielded straight lines with a positive slope, which verified the first-order dependence on the hydroxide ion or deuteroxide ion. The activation energy of the epimerization process was 21.2 kcal/mole. In aqueous solution at high pH, epimerization rather than conversion to ampicillin represents a major pathway of hetacillin degradation, although the beta-lactam ring of the hetacillin molecule is highly resistant to attack by the hydroxide ion.


Actions of mescaline on isolated rat atria. Mescaline, in concentrations of 5 x 10(-4) and 1 x 10(-3) M, produced negative chronotropic and positive inotropic responses in isolated, spontaneously beating rat atria. In tissues driven at a constant rate, the inotropic response was diminished greatly, indicating that the increment in the force of contraction was secondary to the reduction in rate. These chronotropic and inotropic responses were not altered consistently by pretreatment with the histamine antagonists chlorpheniramine and metiamide.


Electronic absorption spectra and protolytic equilibria of doxorubicin: direct spectrophotometric determination of microconstants. The ground- and excited-state dissociation constants and the electronic absorption and fluorescence spectra of doxorubicin were investigated by spectrophotometry. A general method for the direct calculation of individual microscopic dissociation constants was derived using the spectrophotometric data obtained. It was concluded that the protonated amino sugar group is slightly more acidic than the phenolic group. The spectrophotometric data were analyzed, and the macro- and microconstants for the various equilibria was reported.


Solubility and ionization characteristics of phenytoin. The solubility of phenytoin was determined in pH 7.4 and 5.4 phosphate buffers at five temperatures; in hydroalcoholic solutions, 0--4% methanol; and in pH 4.8--8.4 buffer solutions. From the temperature data, the enthalpy and entropy of solution of this nonideal system were calculated and were similar at both pH values. The data obtained from the buffer solutions were used to calculate the apparent dissociation constant, pKa', of phenytoin as 8.06. A GLC method with on-column methylation was used to quantitate phenytoin with 5-(p-methyl-phenyl)-5-phenylhydantoin as an internal standard. The assay uses chloroform of extraction of the drug from aqueous solutions. The ratio of peak heights was adjusted for weights of aqueous and organic layers, and results were calculated in micrograms per gram of sample and mole fraction of phenytoin. Although hydroalcoholic solutions enhanced drug solubility, there is a potentially significant disadvantage in using alcohol for clinical studies.


Determination of acenocoumarol in plasma and urine by double radioisotope derivative analysis. Acenocoumarol, to which a 14C-labeled internal standard has been added, is extracted at pH 4 into ethyl acetate-heptane (20:80 v/v), back-extracted into aqueous sodium hydroxide solution after solvent washing with a pH 7 buffer, and reextracted after acidification in the solvent mixture. It is then acetylated with 3H-acetic anhydride. The acenocoumarol acetyl derivative is separated from the metabolite derivatives by TLC, and its radioactivity is measured. The method is specific and sensitive to a concentration of 8 ng/ml.


Kinetics of accumulation, efflux and the pharmacological effects of tritiated dihydroazapetine on the rabbit aorta. Azepatine, a potent alpha adrenergic antagonist, was catalytically reduced with tritium and hydrogen gas to form dihydroazapetine. The pA2 azapetine was 7.9 whereas that of dihydroazapetine was 6.6, corresponding to a 20-fold decrease in potency. 3H-dihydroazapetine is accumulated into three kinetically distinct compartment in the denervated rabbit aorta. Likewise, efflux of the labeled antagonist occurs from three compartments. The rat constant for onset of alpha adrenoreceptor blockade is 3.15 min-1 which is nearly identical to the rate constant for entry of 3H-dihydroazapetine into a rapidly filling compartment (3.86 min-1) possibly representing the extracellular space. These data are consistent with the hypothesis that onset of alpha adrenoreceptor blockade by dihydroazapetine is diffusion limited.


Differences in pulmonary and cardiovascular beta receptors in the guinea pig and rabbit. An in vivo preparation, in which a body plethysmograph was incorporated, was useful in monitoring the cardiopulmonary effects of pharmacological agents in the guinea pig and rabbit. Isoproterenol, given 30 seconds prior to histamine challenge, reproducibly blocked histamine-induced dynamic compliance decreases and increased heart in the artificially ventilated guinea pig. These effects were used to separate the activity of beta adrenergic blockers on airway and heart muscle. Dose-response data were obtained and ED50 values for pulmonary and cardiovascular blockade were compared. Relative potencies and cardioselectivity ratios for dichloroisoproterenol, practolol, dl-propranolol and d-propranolol were determined. Both practolol and dichloroisoproterenol were cardioselective; dl-propranolol was found to be the most potent. When a similar protocol was tried in the rabbit, isorpoterenol failed to antagonize either histamine or methacholine-induced airway constriction. This finding was supported in subsequent in vitro tests. Isoproterenol and epinephrine were ineffective in blocking methacholine-induced tracheal chain contractions and epinephrine did not significantly enhance adenylate cyclase activity. Our observations suggest rabbit airway smooth muscle is insensitive to beta adrenergic stimulants.


Phosphofructokinase of Leishmania donovani and Leishmania braziliensis and its role in glycolysis. It was shown in an investigation of the phosphofructokinases of Leishmania donovani and Leishmania braziliensis that both enzymes are similar to that of Crithidia fasciculata. Although the enzymes are allosteric with respect to their substrates and require AMP for activation, there is no influence by other heterotropic modifiers. The Mg2+-ATP chelate activates these enzymes in a first order process and they can be inhibited by free ATP. The inhibition is reversed by the activator, AMP, in a competitive manner. The requirement for the nucleotide in L. donovani can be eliminated by decreasing the pH. The data indicate that phosphofructokinase, a pivotal enzyme in glycolysis for most organisms, probably does not play an important role in glycolysis in Leishmania.


Nature of the rabbit acrosome reaction-inducing activity of follicular fluid. Follicular fluid samples from rabbits, cats, pigs, women and cows had acrosome reaction-inducing activity (ARIA) on rabbit spermatozoa as determined by differential staining after incubation with these fluids. Activity was retained after dialysis and at least 50% was found to be labile when heated to 56 degrees C for as little as 20 min. The induction of the acrosome reaction by bovine follicular fluid showed a dependence on the concentration of follicular fluid and spermatozoa and on calcium ions, and had a pH optimum of approximately 8. Enzyme treatments showed that proteases destroyed the ARIA and this activity was completely blocked by treatment of bovine follicular fluid with goat anti-bovine antiserum. Electron microscope observations indicated the similarity of the reactions observed to that occurring in vivo. It is concluded that the rabbit acrosome reaction inducing-activity of bovine follicular fluid is a serum component(s), probably a protein(s) of high molecular weight.


Experimental E. coli arthritis in the rabbit. A model of infectious and post-infectious inflammatory synovitis. Rabbit knee joints challenged with E. coli 06 underwent a self-limited infection lasting several weeks followed by a prolonged post-infectious inflammatory arthritis. The E. coli used did not possess collagenolytic activity nor did a variety of common aerobic clinical isolates. Articular cartilage destruction occurred by two basically different mechanisms: u) direct invasion of pannus at the juxtaarticular margins, and 2) fibrillation in cartilage to cartilage contact areas. Weekly measurement of intra-articular pH and temperature were correlated with bacteriologic findings and groww and microscopic pathologic events.


An analysis of the professional performance of physician's assistants. The job performance of a national sample of 939 physician's assistants has been assessed by means of a self-rating scale as well as by a rating scale completed by supervising physicians. Three-quarters of the participating supervising physicians are greatly satisfied with the work of their physician's assistants and a similar proportion indicate that they would definitely hire the same assistant if they "had it to do over again." The influences of background and work environment characteristics upon performance were assessed by means of multiple regression. Graduating from a military-sponsored physician's assistant program appears to have a beneficial effect upon performance. Performance during training exerts only a weak effect upon job performance. Amount of education and medical experience prior to beginning physician's assistant training do not affect job performance.


Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus. Intact Sindbis virus and Triton-solubilized viral glycoprotein were treated with alpha-mannosidase and with a preparation of mixed glycosidases from Diplococcus pneumoniae to probe the accesibility of carbohydrate units on the viral surface. The products of glycosidase attack on Triton-solubilized virus showed that mose carbohydrate units of the glycoproteins are good substrates for these enzymes. The relative resistance of most of the viral oligosaccharides in intact virus particles showed that much of the carbohydrate is not accessible to glycosidases, probably because it is not exposed at the viral surface. The only completely accessible carbohydrate units on Sindbis glycoproteins were the type A oligosaccharides of E2. This differential accessibility of Sindbis oligosaccharides is discussed in relation to the organization of the viral surface.


Acute purulent otitis media in children older than 5 years. Incidence of Haemophilus as a causative organism. It is generally believed that Haemophilus influenzae is not commonly a causative agent of otitis media in children older than 5 years of age. We recently studied cases of 58 children, aged from 5 to 9 years, who had acute otitis media. Haemophilus species were the causative agents in 36% of cases. This high incidence of Haemophilus isolation from the middle ear exudate of these children has important therapeutic implications; antibiotics effective against H influenzae should be employed when dealing with otitis media in this older age group.


Severe combined immunodeficiency disease. Characterization of the disease and results of transplantation. Pretransplant and posttransplant data for 69 patients with severe combined immunodeficiency disease are presented. Both B and T lymphocyte functions were absent in approximately 80% of the children and markedly depressed in the remainder. Transplantation of marrow from HLA genotypically identical donors provided the highest six-month survival rate (63%); six-month survival rates for patients who received fetal tissue transplants (43%) or marrow from mixed leukocyte culture (MLC) negative donors (38%) were significantly higher (P less than .05) than for patients treated with marrow from MLC positive donors (5%). Additional factors appeared to influence survival and the severity of graft-vs-host (GVH) disease. Patients more than 6 months of age had more intense GVH disease than younger patients. Survival rates  were lower and GVH disease more intense when boys received transplants from girl donors than the reverse.


Nephropathia epidemica. The Scandinavian form of hemorrhagic fever with renal syndrome. We report epidemiologic, clinical, laboratory, and biopsy findings in 14 cases of nephropathia epidemica. The patients were between 19 and 49 years of age. The onset of the disease was characterized by high fever, nausea, headache, backache, abdominal pain, proteinuria, oliguria, hematuria, and uremia. The symptoms subsided rapidly during the polyuria phase, which followed the oliguria stage. Because of renal failure, hemodialysis was required in eight cases. Edema of eyelids, conjunctival injection and hemorrhages, transitory myopia, and acute glaucoma were the most common eye abnormalities. Renal biopsy specimens showed glomerular changes, with mild swelling of the epithelial cells of Bowman's capsule, thickening of the basement membrane of glomerular capillaries, glomerular adhesions, inflammatory cell infiltration, leukocytoclasis and hemorrhages in the interstitium, and eosinophilic hyaline degeneration and vacuolization of the epithelial cells of the proximal tubuli.


Pharmacologic analysis of stretch-induced sinus acceleration of the isolated dog atrium. Using isolated, blood-perfused atrium preparation of the dogs, the effect of stretch on sinus rate was studied in 11 preparations. Stretching usually produced sinus acceleration in all 11 spontaneously beating atria. Within a range of 0--30 Gm, greater degrees of stretch produced greater degrees of sinus acceleration. During maintained stretch sinus rate slightly reduced but not to the control rate. On release from stretch sinus rate immediately returned to the control rate, and there was occasionally a slowing to rates lower than control in verapamil treated preparations. Atropine, adrenergic beta-receptor blocking agents, propranolol and carteolol, verapamil, and tetrodotoxin did not greatly modify responses to stretching.


A comparative study of the effects of OPC-1085 and propranolol on isolated guinea pig atrium. Effects of a newly synthesized beta-blocker, 5-(3-tert-butylamino-2-hydroxy) propoxy-3,4-dihydrocarbostyril (OPC-1085) were compared with those of propranolol. OPC-1085 had a potency about 3 times greater than that of propranolol in blocking the positive inotropic and chronotropic effects of isoprenaline on the isolated guinea pig atrium. At a concentration of over 3 X 10(-5) M OPC-1085 produced negative inotropic and chronotropic effects. However, these effects were about 10 times weaker than those of propranolol. Suppressing effects on the rate of rise and on the maximum driving frequency of action potentials were also more than 10 times less than those of propranolol. There was almost no change in the action potential of vagus nerve after a 10 min treatment with OPC-1085 (10(-5) M), while the action potential was reduced to 60-70% with propranolol (10(-5) M).


Stimulative effects of chelating agents, 2,2'-bipyridine and 1,10-phenanthroline, on lipid peroxidation in rat liver microsomes. Well known lipid peroxidation inhibitors, 1,10-phenanthroline and 2,2'-bipyridine, stimulated microsomal NADPH- and ascorbic acid-dependent lipid peroxidation when low concentrations of these chelating agents were added to incubation mixture. The stimulatory effects of the chelating agents on lipid peroxidation were enhanced when ferrous ion was added together with the chelating agents to the mixture at a molar ratio of 1:1. Ethylenediaminetetraacetic acid (EDTA) had no stimulatory effect on lipid peroxidation. Ferrous ion-EDTA complex increased lipid peroxidation by only 20-30%, which was lower than that obtained by addition of the same concentration of ferrous ion alone. On the other hand, manganese and calcium ions, which are also inhibitors of lipid peroxidation, had no ability to stimulate lipid peroxidation even in the presence of extra ferrous ions. Changes in the lipid peroxidation by chelating agents affected the apparent activity of ethylmorphine N-demethylation.


Analysis of hypotensive mechanisms of pindolol, a beta-adrenoceptor blocking drugs in rats. The hypotensive mechanisms of pindolol in anesthetized and conscious rats were investigated. Pindolol caused a fall in blood pressure in anesthetized, conscious and spinal rats, though in conscious rats a higher dose of the drug was required to produce such a response. This hypotension with pindolol in anesthetized and conscious rats was markedly inhibited by pretreatment with the drug itself or other beta-adrenoceptor blocking drugs. A similar phenomenon also occurred when isoproterenol was injected intravenously in anesthetized and conscious rats. The relationship between the hypotensive actions of four beta-adrenoceptor blocking drugs in anesthetized rats and their intrinsic beta-sympathomimetic actions in isolated catecholamine-depleted tracheal preparations was determined. Order of hypotensive potencies was the same as that of their intrinsic beta-sympathomimetic action, namely, pindolol greater than carteolol greater than bufetolol in equilibrium propranolol (p less than 0.05). These results suggest that the hypotension with pindolol is mediated through a decrease in the peripheral vascular resistance due to an intrinsic beta-sympathomimetic action of the drug.


Drug-induced dystonic reactions.

Drug-induced dystonic reactions. In the 12 unusual cases of drug-induced dystonic reaction discussed, major tranquilizers were ingested unaccompanied by anti-parkinsonian drugs. The five characteristics of the drug-induced dystonic reactions are oculogyric crisis, torticollic crisis, buccolingual crisis, opisthotonic crisis, and tortipelvic and gait crisis. Neuromuscular manifestations occur in the oculogyric, torticollic, buccolingual, opisthotonic and tortipelvic regions. The basic treatment is to discontinue major tranquilizers and administer anticholinergic agents to block the enhanced cholinergic function. Useful anticholinergic and antihistaminic agents for the treatment of drug-induced dystonic reactions are listed to aid in the selection of appropriate therapeutic remedies.


[Ocular disturbances in asthenic neurosis and their treatment (author's transl)]. The author has studied over 200 cases of asthenic neurosis. The ocular manifestations could be classified into four clinical types according to the dominant ocular symptoms. 58.34% of these patients were female and 41.66% were male. 78% of the patients were between 20 and 40 years. 59% were students. In 75% of these cases a raised retinal blood pressure as an ocular manifestation was discovered. The ocular disturbances in asthenic neurosis are present especially in persons who regularly work more than 8 hours continuously. The treatment consists of physical and intellectual rest periods, sedatives, tonics, and connective tissue therapy with extracts of whole eyeballs. In 80% of cases we had favourable results.


Plasma catecholamines in exercise induced bronchoconstriction. Plasma nor-epinephrine (NE) and epinephrine (E) levels at rest and immediately after exercise were estimated in 8 patients with asymptomatic extrinsic allergic bronchial asthma. The patients had a normal airway resistance at rest and developed a marked bronchoconstriction (EIB) during exercise, which could be prevented by previous alpha-adrenergic blockade with phentolamine. In 7 control persons NE and E levels were measured also after beta-adrenergic blockade with propranolol. The following results were obtained: 1. At rest NE levels showed no significant differences between the groups. After exercise an increase of NE was observed in all groups, but in patients, even after phentolamine, and in normals after propranolol the increase was significantly higher than in the normal group within the control test. 2. No significant differences between the groups were found in E levels at rest and after exercise. Exercise caused no significant increase of E levels, except in the normals after propranolol application. 3. No significant correlation existed between NE levels and the increase of airway resistance after exercise. It is concluded that during exercise in asthmatics the sympathetic activity is enhanced, but the provocation of an EIB does not seem to be mediated by enhanced plasma NE levels.


Allergic granulomatosis and angiitis (Churg-Strauss syndrome). Report and analysis of 30 cases. The clinical and morphologic findings of allergic granulomatosis and angiitis of Churg and Strauss in 21 men and 9 women were reviewed. The classic features are those of systemic vasculitis in a setting of bronchial asthma and eosinophilia. Pathologically there is necrotizing vasculitis of small arteries and veins with extravascular granulomas, and infiltration of vessels and perivascular tissues with eosinophilia. These features differentiate it from poly-arteritis nodosa. The lungs, peripheral nerves, and skin are most frequently involved. Renal failure was encountered in only one patient in this series. Shortness of the interval from onset of asthma to appearance of vasculitis is an unfavorable prognostic sign. Corticosteroids seem to influence long-term survival favorably.