Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties. Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable  in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.


N-acetylphenylalanyl-tRNA hydrolase from yeast. Purification and properties. This paper describes the purification and properties of an enzyme present in yeast which splits N-acetylphenylalanyl-tRNA to N-acetylphenylalanine and tRNA. The enzyme has been 35000 as estimated by filtration  on Sephadex G-150, is maximally active in the presence of a divalent cation (Mg2+ , Mn2+ or Ca2+) and has a pH optimum at around neutrality. The enzyme is highly specific in hydrolyzing N-acetylphenylalanyl-tRNA (Km = 0.4 micron). Phenylalanyl-tRNA is hydrolyzed with a similar apparent affinity but with an efficiency of 40% of that found for N-acetylphenylalanyl-tRNA. Other free or N-substituted aminoacyl-tRNAs are not substrates of this hydrolase. Neither of the two reaction products are effective inhibitors of this enzyme. Based on its substrate specificity, the trivial name of N-acetylphenylalanyl-tRNA hydrolase is proposed for this enzyme.


Protons and Mg2+ cations as probes in investigating the role of GTP in initiation complex formation. fMet-tRNAfMet binding to both 30-S subunits and to 70-S particles is dependent on both pH AND Mg2+ concentration: for fMet-tRNAfMet binding to 70-S particles, variations of pH and Mg2+ concentration are tightly interdependent. This behavior can be interpreted by the polyelectrolyte theory as a direct consequence of the fact that the binding occurs in a polyanionic micro-environment. The pH-dependent binding to 70-S particles clearly shows the involvement of two prototropic groups which appear to be those carrying out GTP hydrolysis, therefore directly linked to initiation complex formation; in the presence of a non-hydrolyzable analogue to GTP, guanosine 5'-[beta, gamma-imido]triphosphate, the binding of fMet-tRNAfMet shows much less interdependence between variation of pH and Mg2+ concentration.


Effects of terfenadine and diphenhydramine alone or in combination with diazepam or alcohol on psychomotor performance and subjective feelings. The effects of single oral doses of terfenadine, diphenhydramine and placebo, alone or in combination with diazepam or alcohol, on psychomotor performance and subjective feelings were evaluated in a double-blind, crossover study in 20 normal male volunteers. Terfenadine 60, 120 and 240 mg had no effect on psychomotor skills and subjective feelings, whereas diphenhydramine 100 mg slightly impaired certain features of psychomotor performance and severely worsened subjective feelings. Terfenadine 120 mg did not influence the adverse effects of oral diazepam 10 mg or of alcohol 0.75 g/kg on psychomotor performance and subjective feelings. In contrast, diphenhydramine 100 mg significantly enhanced these effects of diazepam and alcohol.


The influence of pH on the sex-related differences in renal organic ion transport. The stimulating effects of elevated medium pH and androgen on in vitro transport of p-aminohippurate and N-methylnicotinamide (NMN) were additive, although the androgenic effect was pH-dependent only in the case of NMN. The similarity of response of the 2 systems supports the idea of a common passive efflux pathway for organic anions and cations.


Neuroendocrine mechanisms and aging.

Neuroendocrine mechanisms and aging. Evidence describing altered neuroendocrine function during aging from this and other laboratories is reviewed, with focus on changes in the brain-pituitary-ovarian-adrenal-hepatic and in the brain-pituitary-ovarian systems. Difficulties in interpreting the discordant data on age-related changes in pituitary function are discussed. Among mechanisms of reproductive aging are changes at both the ovarian and hypothalamic level (including reduced catecholamine levels, turnover, and synaptosomal uptake). However, it cannot yet be concluded that impairments of hypothalamic catecholamine metabolism are the primary cause for the loss of regular cycles. Evidence for dopaminergic impairments in the basal ganglions of humans and rodents during normal aging suggests that these changes may be a general phenomenon of aging. Although the origins of the changes are not yet known, neuronal cell loss in the substantia nigra would not seem to be the only cause.


Fetal-maternal hemorrhage after amniocentesis: incidence, degree and ramifications. A review of the literature indicates a small but significant incidence of fetal-maternal hemorrhage following amniocentesis. In these studies, consideration has not been given to the frequency with which erythrocytes containing hemoglobin F are to be found in the maternal circulation in the absence of amniocentesis. In this investigation, Kleihauer-Betke analysis was carried out before and after amniocentesis. This study confirms previous reports that spontaneous fetal maternal hemorrhage is not uncommon during normal intrauterine gestation and that amniocentesis is probably only an occasional cause of fetal-maternal hemorrhage. This impression is based on the findings that most Kleihauer-positive maternal blood samples after amniocentesis were also positive before amniocentesis. In spite of these findings, and since Rh-immune serum globulin is apparently harmless to the fetus and mother, its use is still advocated in nonsensitized Rh-negative mothers after amniocentesis.


Repeat laparotomy as a preclinical diagnostic and therapeutic method for treating ovarian carcinoma. One hundred forty-two cases of epithelial ovarian carcinoma were treated from 1972 to 1976 in the Department of Obstetrics and Gynaecology, University Central Hospital, Turku, Finland. Repeat laparotomy was performed on 37 of the patients about six months after total extirpative surgery (group I). In addition, a second laparotomy was performed on 14 patients after partial surgery or exploratory laparotomy (group II). All patients were given external radiation therapy combined with cytostatic treatment prior to the repeat laparotomies. A preclinical carcinoma was detected in six of the 37 patients in group I. Two of them are asymptomatic after five years of follow-up. In group II, total surgery after initial partial or exploratory laparotomy succeeded in half of the cases. Serous and endometroid carcinomas appeared to respond best to conservative therapy preceding repeat laparotomy.


Surgical closure of the incompetent cervix: 15 years' experience. This paper presents the author's experience, over a 15-year period, with 33 patients who demonstrated cervical incompetence and who were treated by surgical procedures. The surgical approach is unique in that the band is placed under direct vision above the uterosacral ligaments and through the cardinal ligaments. Slippage of the band, with resultant loss of pregnancy, is most unlikely. Fifty-three pregnancies occurred in this group. Each patient treated by the described technique produced one or more living children. Complications which occurred in the group are also discussed.


The effectiveness of obligatory lymphadenectomy in treating carcinoma of the cervix. For the past 20 years, obligatory lymphadenectomy has been performed with the Wertheim's operation for carcinoma of the cervix at the First University Women's Clinic in Vienna. Formerly, selective lymphadenectomy was performed with the radical operation, with only nodes suspected of metastasis being removed. The theoretical advantages and disadvantages of each system are discussed, and several previous reports are analyzed and criticized. Our experience at the First University Women's Clinic shows that rates of mortality from recurrence were higher for a group of patients who underwent selective lymphadenectomy from 1946 to 1949 than for two more recent series who were treated with obligatory lymphadenectomy. Thus, the effectiveness of performing the latter procedure with the Wertheim's operation to reduce mortality from recurrence is confirmed.


Reducing the morbidity of vacuum aspiration abortion. This paper reports an analysis of the measures taken at Preterm, Inc, of Washington, DC, to reduce the incidence of complications of first trimester outpatient abortions. Data are presented on the 291 complications (40.0/1000) resulting from 7272 procedures performed during 1976. Forty-one women were hospitalized (5.6/1000). It has been our experience that routine use of ultrasonography when gestational age is unclear, careful examination of tissue especially when a small quantity is present and early use of reevacuation to diagnose and treat postabortal complications are essential in reducing the incidence and severity of abortion complications.


Consumption coagulopathy associated with intrauterine fetal death: the role of heparin therapy. A gravida with intrauterine fetal death who developed progressive chronic consumption coagulopathy was treated with heparin. When serial fibrinogen levels fell below 100 mg% and the prothrombin time was significantly prolonged, intravenously injected heparin corrected hypofibrinogenemia. A safe delivery followed administration of oxytocin. The authors emphasize the infrequent need for heparin therapy in the majority of cases of the intrauterine fetal death syndrome. Therapeutic guidelines for its use in selected cases are reviewed.


Successful pregnancy following myocardial infarction: a case report. A case of successful pregnancy following myocardial infarction is reported. The literature on this subject is reviewed and discussed. It is concluded that, if sufficient time is allowed for healing, pregnancy following myocardial infarction is associated with little or no maternal or fetal mortality.  A plan of obstetrical management is outlined.


Histologic malignancy grading in invasive squamous cell carcinoma of the vulva. A preliminary report on a histologic malignancy grading of vulvar carcinoma is presented. A retrospective histologic study of 40 vulvar carcinoma cases stage I and II (TNM-system) with a minimum five-year follow-up was carried out and correlated to the course of the disease. Morphologic criteria characterizing the tumor cell population, as well as the tumor-host relationship, were examined and scored. The scores obtained could be divided into three groups that correlated well with the clinical outcome. The low-score group had no metastases or recurrence, whereas 82% of the high-score group had both metastases and fatalities. Depth of invasion was found to have a strong correlation to clinical outcome. A more accurate morphologic malignancy grading of such carcinomas could lead to a more individual and often less radical treatment plan.


Correlates of human umbilical cord length. In this study of the umbilical cord lengths of 602 Nigerian infants delivered at term, the mean, median and modal measurements were 57.48, 57.71 and 68.00 cm, respectively (range = 20-100 cm). There was a significant positive correlation between cord length and fetal weight (r = 0.2177, p less than 0.001) and also between cord length and placental weight (r = 0.2504, p less than 0.001). Other fetal and maternal variables studied did not correlate with cord length. Further investigations  of the factors responsible for the growth of the umbilical vessels are needed.


Treating dysmenorrhea with anti-inflammatory agents: a double-blind trial with naproxen sodium. Thirty-two dysmenorrheic patients participated in a double-blind trial of naproxen sodium for three consecutive menstrual cycles. The women were divided into two groups: 15 women were given naproxen sodium (the sodium salt of d-2-(6-methoxy-2-naphthyl) propionic acid) and 17 women received placebo tablets. The women were prescribed two tablets (550 mg) at the first sign of menstrual pain and one tablet (275 mg) thereafter every six hours, as required. There were no significant differences between the two groups in physical characteristics, obstetric and gynecologic histories, including the character of dysmenorrhea and pretreatment pain intensity scores (p = 0.7). Following intake of the drug or placebo, the participants rated the relief provided by the medication with a six-point scoring system. When the scores for pain relief were tallied for the three treatment cycles, the naproxen sodium group averaged 13.7 +/- 0.65 standard error, while the placebo group averaged 8.8 +/- 0.95 standard error out of a possible maximum relief score of 18. The difference between the two groups was statistically significant at p = 0.0004. Few patients reported side effects.


Risks and benefits of culdoscopic female sterilization. This paper reviews the risks and benefits of the culdoscopic approach to female sterilization in an alnalysis of 2153 culdoscopic sterilization procedures performed at 11 centers in nine countries. Inability to occlude the tubes as planned was reported for approximately 6.0% of the cases including 1.4% in which one or both tubes could not be occluded by any technique. Surgical difficulties were reported for about 13.0% of the procedures, and surgical complications, of which the most frequent was torn or bleeding tubes, occurred in 2.0% of the cases. Pregnancy rates were significantly higher for patients whose tubes were occluded by tantalum clips (7.7 per 100 women at 12 months), indicating that this is not the preferred technique of tubal occlusion; the 12-month life table pregnancy rates were 0.1 per 100 women for tubal ring and 0.0 for Pomeroy and fimbriectomy. Comparison of these data with similar pooled data on laparoscopy and minilaparotomy indicates that culdoscopy is associated with greater technical difficulty and morbidity. Thus, abdominal procedures will continue to be preferred for use in most large-scale programs.


Collagenolytic activity and steroid levels after administration of dehydroepiandrosterone sulfate. Seventy-five pregnant women in the 38th to 41st weeks of gestation were given a single intravenous injection of 200 mg of dehydroepiandrosterone sulfate (DHAS). The changes in estriol, 17beta-estradiol and progesterone levels in the serum, the uterine cervix and the myometrium of the placenta-implanting site were then determined. Estriol levels remained unchanged both in serum and tissue, but the level of 17beta-estradiol increased sharply both in serum and tissue after four hours. The increases of 17beta-estradiol in the serum and the portio vaginalis of the same patient were well correlated (r = 0.79898), but the percentage of increase was much higher in the portio vaginalis than in the serum (p less than 0.001). Serum progesterone levels did not change initially, but always decreased within four or eight hours in cases in which labor had started or delivery was accomplished within 24 hours (p less than 0.01 at four hours). The total amount of collagenase was determined in ten subjects before and after the administration of DHAS. The total collagenase activity was elevated by an average of 152.3%. The peak of activity was accelerated from the fourth to the second day (p less than 0.001) of the culture. A probable mechanism of DHAS action in accelerating cervical ripening is presented.


Midtrimester abortion with prostaglandin and hypertonic saline--a comparative study. This study was conducted in Baroda, India, to evaluate and compare the safety and effectiveness of intraamniotic prostaglandin F2alpha (PGF2alpha) and 20% saline augmented with intravenous oxytocin for terminating 200 pregnancies of 14 to 20 weeks' gestation. While there was no method failure among the group treated with saline, ten method failures were reported for patients treated with the PGF2alpha. The rate of incomplete abortions was significantly lower for the group treated with saline (19.4%) than it was for the group treated with the PGF2alpha (33.7%). The administration of oxytocin after fetal expulsion did not reduce the rate of incomplete abortion. The mean instillation-to-abortion time was significantly lower with saline than with PGF2alpha. The incidence of gastrointestinal side effects and excessive bleeding (less than 200 ml) during the procedure was significantly higher for patients treated with PGF2alpha than for those treated with saline.


Ovarian and peripheral venous blood prostaglandin F2alpha levels in pregnant and nonpregnant women. Prostaglandin F2alpha (PGF2alpha) was measured by radioimmunoassay in human blood plasma collected from both ovarian veins and from a peripheral vein in eight pregnant and seven nonpregnant women. In the pregnant women, significantly higher levels of PGF2alpha were found in venous blood plasma taken from the ovary which contained the corpus luteum than were found in either the ovarian vein blood plasma from the opposite ovary or in the blood plasma from a peripheral vein. Thus, PGF2alpha levels in the venous blood plasma from the ovary without a corpus luteum were not significantly different from those found in the peripheral venous blood plasma. In seven nonpregnant women, significantly higher levels of PGF2alpha were found in the venous blood plasma taken from the ovary which contained a corpus luteum than were found in the blood plasma taken from a peripheral vein. The former levels did not differ significantly from those found in the venous blood plasma taken from the ovary without a corpus luteum in these nonpregnant women. Thus, in nonpregnant women a significant difference was found between PGF2alpha levels in the venous blood plasma from the ovary without a corpus luteum and those in peripheral venous blood plasma.


Oesophageal function in cirrhotic patients undergoing injection sclerotherapy for oesophageal varices. Oesophageal function has been studied in three groups of cirrhotic patients: those without varices, those with varices and those with varices treated by injection sclerotherapy. Using the Honeywell Model 31 oesophageal motility probe and the Ingold combined stomach pH electrode, measurements were made of the lower oesophageal sphincter (LES) pressure and length, swallowing responses, reflux and clearance of acid. The presence of varices was associated with an increase in LES length and reduced lower oesophageal contraction pressure during swallowing and some failure of sphincter relaxation during swallowing. Sclerotherapy was associated with a reduction in the maximum LES pressure both at rest and during swallowing, and an impairment of acid clearance, but postural reflux of acid was not observed in any patient.


Oxygen availability from the blood and the effect of phosphate replacement on erythrocyte 2,3-diphosphoglycerate and haemoglobin-oxygen affinity in diabetic ketoacidosis. Eleven patients with diabetic ketoacidosis were given intravenous phosphate in doses (mean 118 mmol; range 83--320 mmol) adequate to maintain normal plasma phosphate, in addition to a standard treatment regime. Prevention of hypophosphataemia stimulated recovery of the initially low red-cell 2,3-diphosphoglycerate concentrations (10.6 +/- 5.8 (SD) mumol/g Hb) after twenty-four hours. In ten control patients (initial concentration 8.1 +/- 4.4 mumol/g Hb) treated without  phosphate replacement, significantly lower red-cell 2,3-diphosphoglycerate concentrations were found between 2 and 6 days after admission (forty-eight hour value for control patients 14.6 +/- 1.6 and for phosphate-treated patients 18.9 +/- 4.1 mumol/g Hb; p less than 0.01). However, no effect on in vivo p 50 or on the availability of oxygen from the blood resulted from the higher 2,3-diphosphoglycerate levels. Maintenance of normal plasma phosphate levels by intravenous phosphate is, therefore, not indicated to improve tissue oxygenation in diabetic ketoacidosis.


On the relationship between gastric pH and pressure in the normal human lower oesophageal sphincter. The effect upon the lower oesophageal sphincter (LOS) of acid in the proximal stomach was investigated in 12 young healthy subjects. The cardia was perfused in turn with solutions at pH 7, 4, 3, 2, and 1, during successive withdrawals of three perfused pressure-recording catheters, openings of which were at the same axial level. Although there was considerable individual variation, there was a direct and significant relationship between LOW pressure and the acidity of the solution perfused. The results suggest that resting LOS pressure and the acidity of the solution perfused. The results suggest that resting LOS pressure may be determined in part by the pH of the gastric content.


The effect of pH on colchicine conformation and structure. The effect of various pH values between 0 and 14 on the structure and conformation of colchicine was examined using UV-vis spectrophotometry at a concentration of 1.7 x 10(-5) M and NMR techniques at a colchicine concentration of 0.1M. The complete interpretation of the colchicine NMR spectrum in D2O is given. A stable structure of the colchicine molecule in aqueous solutions at pH from 2 to 12 was demonstrated. However, during incubation at 40 degrees C colchicine was found to be stable only at pH values between 2 and 10. The significance of these data for reactions of cholchicine in regard to metabolism and interaction with macromolecules is discussed.


Beta-lipotropin and endorphins: implications of current knowledge. Evidence that the endorphins, metabolites of the pituitary hormone beta-lipotropin, profoundly affect mood and behavior, are secreted with ACTH in response to stress, and may act as neurotransmitters in the GI tract, suggests that they play an important role in CNS homeostasis. The fact that these peptides are synthesized in the GI tract as well as the brain casts new light on CNS-endocrine-environmental interrelationships.


The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro. Cyclophosphamide (CY) was activated in vitro with washed rat liver microsomes and cofactors. Pretreatment of mouse spleen cells in vitro with the activated drug abolished their capacity to give a primary antibody response to SRBC and levan on transfer to irradiated syngeneic recipients. However, responsiveness returned if challenge was delayed for 7 or more days after transfer. Part of this was shown to be of donor origin by an allotype marker. The treatment of normal spleen cells with activated CY in vitro also prevented B cells from regenerating their immunoglobulin receptors after capping with anti-immunoglobulin serum. The induction of suppression required contact between lymphocytes and activated CY for at least 30 min at 37 degrees and did not appear following incubation for 1 h at 0 degrees. Since the antibody response of drug-treated spleen cells to SRBC could not be restored with purified normal B or T cells, it is probable that B and T lymphocytes are both susceptible to suppression by activated CY in vitro. Similar pretreatment abrogated the graft-versus-host (GVH) reactivity of spleen cells as measured by survival and in a popliteal lymph node assay. B cell chimerism in F1 recipients of drug-treated parental spleen cells was demonstrated by the presence of congenic allotype markers. This suggests a possible approach for the attenuation of GVH disease which is associated with bone marrow transplantation in man.


Effect of leukocyte hydrolases on bacteria. IX. The release of lipoteichoic acid from group A streptococci and from Strep. mutans by leukocyte extracts and by lysozyme: relation to tissue damage in inflammatory sites. Human leukocyte extracts, egg white lysozyme, cationic proteins, polymyxin, colimycin, and phenol are capable of releasing lipoteichoic acids (LTA) from group A streptococci and Strep. mutans. While the extraction of LTA by phenol is optimal at pH 4.7, the release of LTA from streptococci by the other agents is optimal at pH 7.4. LTA released by all agents was found to have the same sensitizing abilities, as determined by passive hemagglutination, and to have a similar chemical composition, as shown by thin-layer chromatography and radioactive scanning. The LTA-releasing capacity of all the agents is strongly inhibited by normal human serum. The possible role played by LTA released by leukocyte factors in the pathogenesis of tissue damage during bacterial infections is discussed.


Effect of leukocyte hydrolases on bacteria. X. The role played by leukocyte factors, cationic polyelectrolytes, and by membrane-damaging agents in the lysis of Staphylococcus aureus: relation to chronic inflammatory processes. A heat-stable factor present in extracts of human blood leukocytes is capable of lysing young Staphylococcus aureus at pH 5.0. Lysis is characterized by breakdown of cell-wall components as judged by electron microscopic and biochemical analysis. The leukocyte extracts can be replaced by a variety of agents known to injure cell membranes, e.g., leukocyte cationic protein histone, polymyxin B, colimycin, phospholipase A, and lysolecithin. The mechanisms by which all these agents bring about the degradation of the staphylococcal walls was studied. By using 14C-labeled cell walls devoid of cytoplasmic structures, it was demonstrated that none of the above-mentioned agents had a direct lytic effect on purified cell walls. On the other hand, when any of these agents first interacted with intact staphylococci, a factor (presumably an autolysin) was generated that directly lysed the cell walls. Lysis of cell walls in the presence of intact staphylococci used as a source of autolysin was strongly inhibited by a variety of anionic polyelectrolytes such as heparine and liquoid. The possible role played by bacterial autolysins in the generation of microbial cell-wall components capable of triggering chronic inflammation is discussed.


Cerebrovascular effects of YC-93, a new vasodilator, in dogs, monkeys and human patients. Cerebrovascular effects of YC-93, a new 1,4-dihydropyridine derivative, was examined in experimental animals and in human patients using a variety of methods. YC-93 in doses ranging from 0.001 to 0.03 mg/kg i.v. increased regional cerebral cortical blood flow and cerebral venous outflow in anesthetized dogs, and internal carotid blood flow in anesthetized monkeys. The increase in cerebral blood flow was accompanied by an increase in oxygen delivery to the brain and an elevation of cerebrospinal fluid pressure. Both intracarotid injection and intraduodenal administration of YC-93 also resulted in cerebral vasodilation in monkeys. In patients with cerebrovascular diseases and other chronic diseases, measurements of cerebral blood flow by 133-xenon clearance method showed that an intracarotid injection of 1 microgram/kg of YC-93 increased cerebral blood flow by 28.8% without changing arterial blood pressure and arterial pCO2, and that an intravenous injection of 0.01 mg/kg of YC-93 increased cerebral blood flow by 17.0% with a minimal decrease in the arterial blood pressure but without changing arterial pCO2. Thus, YC-93 produced a potent cerebral vasodilation not only in experimental animals but also in human patients in the same dose, and seemed to act perhaps directly on the cerebral vascular beds.


Potentiation of the mydriatic effect of norepinephrine in the rabbit after monoamine oxidase inhibition. Dose-response curves of pupillary dilation after topical administration of norepinephrine or methoxamine have been determined in rabbits after chronic inhibition of ocular monoamine oxidase by treatment with pargyline or pheniprazine. Eyes treated with either monoamine oxidase inhibitor showed an enhanced responsiveness to the mydriatic effect of norepinephrine given either topically or intravenously. Increments in pupil size of the treated and control eyes in response to methoxamine applied topically, on the other hand, were the same. These results suggest that monoamine oxidase may play a role in the iris as one factor influencing the concentration of norepinephrine at the receptors.


Study on malnutrition. III. Biochemical assessment of the dietary treatment and evolution of the illness. During the course of a malnutrition study, the efficiency of two diets has been followed by clinical observations and biochemical tests. The diets were adapted to the physiological state of 25 patients (16 children, 6 young mothers, 1 adolescent and 2 men) undergoing treatment in a rural hospital at Yasa-Bonga, Zaïre. One group of patients was examined after two weeks of treatment (6 children, 2 mothers), the other group after two months (8 children, 5 adults). In order to study the evolution of the illness, 9 children and 3 young mothers were examined regularly after the return to their villages every 6 months for 2-4 years. The patients responded positively to both diets. After two months of treatment they had clinically recovered, except for the most serious cases. After two weeks of treatment a deficiency in electrolytes, low levels of prealbumin and a net increase in transferrin were noted. After 2 months of treatment the children had regained their normal growth (hydroxyproline index), and most of the biochemical parameters had reached their normal value despite a few deficiencies in electrolytes, phosphorus and magnesium. However, the prealbumin level remained low, particularly amongst children suffering from relapses. In a few cases the activities of cholinesterase (CHE) and gamma-glutamyltranspeptidase (gamma-GT) remained low, which was taken as a sign of poor response to treatment. Generally, the adults responded more slowly to treatment than the children. Regular examinations carried out on certain patients on their return to the villages permitted 2 groups to be distinguished: the first one was composed of patients recuperating well due to good social conditions; the second group of patients suffered from relapses due to lack of hygiene and of rudimentary knowledge of nutrition, and above all severe social problems. The condition of the liver of all these patients was very important; it could be estimated by the determination of the serum levels of prealbumin and the activities of CHE, gamma-GT and isoenzymes of gamma-GT.


On the product of the reaction between cysteamine and 3-bromopyruvate. Some properties of TZCA, the addition compounds of cysteamine and 3-bromopyruvate, have been investigated. From the behaviour of the UV absorption spectra in acidic and alkaline solutions in the presence or absence of oxygen, it was shown that the instability of TZCA was imputable to an oxidative degradation. It was further shown that TZCA undergoes in alkali spontaneous oxidative decarboxylation, and that the arising product may be hydrolyzed to cystamine and glyoxylic acid. Some chemical reactions and the paper chromatographic behaviour of TZCA are reported. It was shown that TZCA, despite its great instability, may be the reactions described, and thus differentiated from other adducts of bromopyruvate and different aminothiols.


Electroncytochemical and biochemical demonstration of guanylate cyclase activity in the pancreatic islet. With a cytochemical method using guanylyl imidodiphosphate as a substrate, the guanylate cyclase activity was localized on the plasma membrane of A, B and D cells of islets of Langerhans isolated from the rat. Adequate control experiments were performed by a double-blind method. Parallel biochemical assay showed that guanylate cyclase activity was not completely lost after fixation with 1% glutaraldehyde and incubation with 4 mM lead nitrate. Furthermore, the depressed activity was still stimulatable with acetylcholine.


Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans. The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.


Metabolism of phenol and resorcinol in Trichosporon cutaneum. Trichosporon cutaneum was grown with phenol or resorcinol as the carbon source. The formation of beta-ketoadipate from phenol, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of phenol begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to beta-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to beta-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2-oxygenase, phenol hydroxylase, and muconate lactonizing enzyme on a diethyl-aminoethyl-Sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizing enzyme. Methyl-, fluoro-, and chlorophenols are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate, beta-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of phenol and vice versa.


Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae. A method is shown to be effective over a wide range of enzyme ratios for the simultaneous detection of the two isoenzymes of acetyl coenzyme A synthetase [acetate:coenzyme A ligase (AMP-forming); EC 6.2.1.1] in homogenates and cellular fractions of Saccharomyces cerevisiae. When this method was used, it was found that cells grown under anaerobic conditions contained only one variety of this enzyme, designated the nonaerobic synthetase, whereas cells grown with vigorous aeration contained principally the other, aerobic, synthetase. In cells grown as standing cultures (i.e., semi-aerobically), both enzymes were present and were found mainly in the extramitochondrial material of homogenates. When anaerobic cultures were aerated, the amount of aerobic enzyme increased steadily over a 24-h period, so that at the end of this time, aerated cells contained predominantly aerobic enzyme. During this same period, the amount of nonaerobic enzyme decreased. The percentage of aerobic enzyme that sedimented with the mitochondria increased steadily during this period of aeration, so that, at the end of 24 h of aeration, essentially all of the aerobic enzyme sedimented with the mitochondria. The nonaerobic enzyme was never found in this cellular compartment.


Tumor inhibitory and non-tumor inhibitory L-asparaginases from Pseudomonas geniculata. Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.


Transport of coenzyme M (2-mercaptoethanesulfonic acid) in Methanobacterium ruminantium. A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.


Mannosyl transfer by membranes of Aspergillus niger: mannosylation of endogenous acceptors and partial analysis of the products. A smooth membrane fraction of Aspergillus niger catalyzed the transfer of mannose from GDP-mannose to endogenous lipid and protein acceptors. The mannolipid was acidic, as judged by diethylaminoethyl-cellulose chromatography, and had a mobility similar to ficaprenyl phosphate on thin-layer chromatograms. Mannose transfer occurred optimally at pH 6.5 to 7.5 and required Mn(2+) for use of the protein as acceptor, but either Mn(2+) or Mg(2+) with the lipid as acceptor. Glycopeptides of the mannosylated protein ([(14)C]gly) and of an alpha-glucosidase (alpha-glu) secreted by the organism were produced by Pronase digestion and separation of the products on Sephadex G-25. Because ovalbumin has a carbohydrate composition similar to that of alpha-glu and because the carbohydrate structure of ovalbumin is known, ovalbumin glycopeptides (Ov) were similarly obtained and used as standards in determining carbohydrate structures. Oligosaccharide chains of [(14)C]gly, alpha-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-beta-N-acetylglucosaminidase, reduction with NaBT(4), and concanavalin A-Sepharose chromatography. The (3)H-labeled oligosaccharides obtained were subjected to the following treatments: (i) digestion with alpha- and beta-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, alpha-glu was permethylated, and the partially methylated alditol acetates were analyzed by gas-liquid chromatography. The resultant proposed structure shows that the oligosaccharide chain of alpha-glu is almost identical to that of an Ov chain, while [(14)C]gly has a structure which is probably the same as that of alpha-glu. It is suggested that the transferase(s) involved in [(14)C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.


Genetic analysis of Staphylococcus aureus RNA polymerase mutants. Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.


Regulation of fatty acid biosynthesis by hydrocarbon substrates in Mycobacterium convolutum. When Mycobacterium convolutum R22 was grown on the n-alkanes C13 through C16, the predominant fatty acids were of the same chain length as the growth substrate. Cells grown on C13 through C16 n-alkanes incorporated between 15 and 85 pmol of acetate per microgram of lipid into the fatty acids, whereas acetate- or propane-grown cells incorporated 280 and 255 pmol of acetate per microgram of lipid, respectively. In vivo experiments demonstrated that hexadecane, hexadecanoic acid, and hexadecanoylcoenzyme A (CoA) all inhibited de novo fatty acid synthesis. Hexadecanoyl-CoA was the most potent inhibitor. Hexadecane and hexadecanoic acid inhibited acetyl-CoA carboxylase by up to 37 and 39%, respectively, at 1 mM. Hexadecanoyl-CoA inhibited the enzyme activity by 65% at 50 micrometer. Cells that were grown on C14 through C16 n-alkanes had about 25 times less acetyl-CoA carboxylase activity than did cells grown on acetate or propane, suggesting repressed levels of the enzyme. Hexadecane- or pentadecane-grown cells were found to have 5 to 10 times more intracellular free fatty acid than cells grown on acetate, propane, or ethane.


Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum. Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.


Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis. beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.


Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction? Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.


Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus. Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.


Long term treatment of tardive dyskinesia. A 4 year trial to treat tardive dyskinesia by a very slow progressive stepwise diminution of the neuroleptic dose and of antiparkinsonian agents as well as by administration of small, slowly increasing and then decreasing doses of reserpine or haloperidol was conducted on 62 chronic schizophrenic patients. This treatment program caused disappearance of tardive dyskinesia in 23, improvement in 26 patients and had no effect in 13 patients. The group of patients with disappearance of tardive dyskinesia had a mean age significantly lower than other groups. The rationale of this treatment was based on the concept of "desensitization" by a slow, progressive unblocking of dopaminergic receptor sites. Another tardive neuroleptic side effect, the "rabbit syndrome" was successfully treated in 7 other patients by antiparkinsonian drugs.


Antipsychotic drugs and seizures.

Antipsychotic drugs and seizures. The authors examine the clinical problem of which antipsychotic drug to use when antipsychotics are indicated in patients with a seizuire disorder or who are susceptible to seizures. While definitive answers to this problem are still unknown, guidelines are offered for antipsychotic drug use in this situation, based on the author's understanding of psychotropics and epilepsy.


Transport of nicotinamide adenine dinucleotide in an unc mutant of Escherichia coli. The presence and some properties of an NAD+ transport system were examined in PA5, a Mg, Ca-ATPase [EC 3.6.1.3]-defective mutant strain of Escherichia coli W2252. NAD+ uptake was stimulated by exogenous energy sources and dependent on external substrate concentrations with an apparent Km of about 25 micrometer. Most of the radioactivity from [14C]-NAD+ accumulated in the cells was identified as NAD+. [14C]NAD+ uptake was competively inhibited by unlabeled NAD+, NADP+, NMN+ or nicotinamide. Similar uptake activity was also observed in W2252.


Lifetime of the purple intermediate of D-amino acid oxidase under anaerobic conditions. The lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral D-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.


A fluorescence study of the effects of pH and Mg2+ on the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. 2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.


Phospholipid-deacylating enzymes of rat small intestinal mucosa. 1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.


Kinetic study of soybean beta-amylase. The effect of pH. 1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.


Chemical modification of epsilon-amino groups in glutamine synthetase from Bacillus stearothermophilus with ethyl acetimidate. The activity of glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus decreased slightly on modification with ethyl acetimidate. Acetamidination of 25--26 of the 2 epsilon-amino groups/subunit of the enzyme affected the maximum velocity, but not the Michaelis constant. The thermostability of the enzyme was considerably increased on acetamidination. Acetamidination of the enzyme did not affect the circular dichroism, the tryptophan fluorescence or the quenching effects of KI and acrylamide on the tryptophan emission. The fluorescence spectrum of p-toluidinylnaphthalene sulfonate bound to the enzyme changed on acetamidination.


Glucagon regulation of protein phosphorylation. Identification of acetyl coenzyme A carboxylase as a substrate. A hormonally induced change in the covalent phosphorylation state of several enzymes is generally regarded as an important mechanism for hormonal modulation of enzyme activity. We have previously demonstrated that epinephrine stimulates the phosphorylation of a peptide of Mr = 220,000 in adipocytes. Incubation of 32P-labeled cytosolic proteins from adipocytes and hepatocytes with antisera raised against homogeneous chicken and rat liver acetyl coenzyme A carboxylase results in the specific and complete precipitation of the same phosphopeptide. No other major phosphopeptide is specifically precipitated. In hepatocytes, glucagon stimulates the incorporation of 32P into this peptide associated with an inhibition of enzyme activity. These data, coupled with previous studies in adipocytes, suggest that cyclic AMP-dependent protein phosphorylation plays a major role in the regulation of acetyl-CoA carboxylase activity and of fatty acid biosynthesis in adipose tissue and liver.


Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis. The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.


Mixed function oxidases in sterol metabolism. Separate routes for electron transfer from NADH and NADPH. Oxidative deformylation of 4-hydroxy[14C]methylene-5alpha-cholest-7-en-3-one and oxidative demethylation of [30,31-14C]4,4-dimethyl-5alpha-cholest-7-en-3beta-ol by rat liver microsomes have been compared with regard to the manner in which electrons are introduced from both NADH and NADPH. Evidence suggests that NADH and NADPH support oxidation of both substrates via separate routes of electron transfer. Thus, 10 micron cytochrome c will inhibit NADPH-supported oxidation to 40 to 50% of control activity leaving NADH-supported oxidation unaffected. Also, treatment of microsomes with subtilisin diminishes NADPH-supported oxidation to 10 to 30% of control activity for either substrate to 70 to 90% of control activity while NADH-supported oxidative activity is virtually unaffected. Studies on the oxidase activities and NADPH-cytochrome c reductase as well as NADH-ferricyanide reductase have shown marked differences in activity in the presence of inhibitors. Thus, 9 mM 2'-AMP inhibits NADPH-cytochrome c reductase to 10 to 20% of control activity while NADPH-supported oxidative demethyl ation and deformylation are essentially unchanged. Mersalyl at 15 to 25 nmol/mg of microsomal protein inhibits both reductases to 20 to 40% of control activity; oxidative demethylation is unaffected and oxidative deformylation stimulated slightly when NADPH is used. Finally, antibody to NADPH-cytochrome c reductase inhibits oxidase activity for either substrate to 70 to 90% of control activity while reductase activity is inhibited to 10 to 30% of control activity.


In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase. We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.


Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein. A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent marine coelenterate, Renilla reniformis, has been purified by conventional methods. One kilogram of processed animals yields approximately 2.7 mg of pure protein with an overall yield of 55%. Physicochemical studies show that BP-LH2 is a globular protein containing one single polypeptide chain with one disulfide bond. Ultracentrifugation studies, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of 4.3. The acidic nature of the protein was confirmed by amino acid analysis, which showed that 27% of the residues are acidic. The protein contains no carbohydrate, phosphate, or tryptophan. There is one noncovalently bound molecule of coelenterate type luciferin resulting in distinct protein spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 = 1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at 520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with Renilla luciferase to give the characteristic in vitro blue bioluminescence. Ca2+ binding produces a distinct change in the spectral properties of BP-LH2 including a 4-fold enhancement of tyrosine fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In addition, the visible absorption maximum shifts from 446 nm to 420 nm. The fluorescence enhancement at 320 nm occurs over the range from 1 to 10 micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of 0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several well studied Ca2+-binding proteins and was found to possess similar Ca2+-binding and physicochemical properties. This study clearly demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in response to an intracellular Ca2+ transient.


Identification of the prosthetic group of urocanase. The mode of its reaction with sodium borohydride and of its photochemical reactivation. Urocanase from Pseudomonas putida and from beef liver were isolated by modifying described procedures. Both enzymes were inactivated and labeled on treatment with tritiated sodium borohydride and gave, upon subsequent hydrolysis, a radioactive acid. The previously reported identity of this acid as 2-hydroxybutanoic acid was disproved by several criteria. Other hydroxy acids were also proved to be different from the radioactive acid derived from urocanase. A large portion of the radioactive material from P. putida was found to be nicotinic acid by 1H NMR spectroscopy, gas-liquid chromatography of its methyl ester, and co-crystallization with authentic reference compounds both as the acid and as the hydrazide. A significant portion of the radioactive material derived from beef liver urocanase also co-crystallized with nicotinic acid. Sodium borohydride-treated inactive urocanase was partially reactivated by light. The action spectrum of the photoreactivation showed a maximum at 330 nm. Treatment of urocanase with sodium borodeuteride followed by hydrolysis afforded a sample of nicotinic acid which carried deuterium mainly in position 6. Both the reversible reducibility of urocanase and its action spectrum of photoreactivation suggest that urocanase contains an enzyme-bound nicotinamide nucleotide molecule which is essential for enzymic activity.


Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli. Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.


A comparison by 220-MHz NMR of histidine hydronium ion titrations in porcine pancreatic ribonuclease and an extensively deglycosylated derivative. 220-MHz NMR was used to observe the titration behavior of the 5 histidine residues in porcine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 3.1.4.22) and a derivative prepared by removal of 80% of the attached carbohydrate from this glycoprotein. Resonances due to histidine C-2 protons were observed over the full pH range for 3 of the residues; such resonances for the remaining 2 histidine residues broadened out as the pH was increased. Resonances due to histidine C-4 protons were also observed for 2 of the residues. The titration curves for both proteins were identical within experimental error. Resonances were assigned by comparison with histidine NMR titrations in ribonucleases from other species. Histidine 105, immediately adjacent to the site of attachment of a heterosaccharide side chain, has a C-2 proton chemical shift and pK that are insensitive to the large alteration in the bulk of the carbohydrate side chain. The chemical shifts of the C-2 proton of histidine 48 and of the C-4 proton of histidine 80, histidine residues that are close to one another and to another heterosaccharide side chain, show a similar insensitivity. The observations are direct evidence in support of the thesis that the heterosaccharides in porcine ribonuclease project away from the surface of the protein into the solution environment.


Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity. A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.


Comparison of the association and orientation of gamma-glutamyltranspeptidase in lecithin vesicles and in native membranes. gamma-Glutamyltranspeptidase purified following solubilization with Triton X-100 can associate with single-layered [14C]lecithin vesicles. Enzyme activity and radiolabeled vesicles were shown to co-migrate during Sepharose 4B chromatography and isopycnic sucrose gradient centrifugation. The enzyme-vesicle complex exhibits a density corresponding to that of a single enzyme molecule bound to a single vesicle, gamma-Glutamyltranspeptidase purified following a solubilization with papain does not bind to vesicles. In addition, papain treatment of vesicles containing the Triton-purified transpeptidase results in the release of 95% of the transpeptidase activity without release of internally trapped [3H]sucrose. The released transpeptidase is chromatographically identical to the papain-purified transpeptidase. gamma-Glutamyltranspeptidase activity associated with both native membranes and with lecithin vesicles exhibits a temperature-induced transition in its energy of activation. In contrast, the proteolytic- and detergent-solubilized forms of the enzyme exhibit a single energy of activation over the entire temperature range. These results suggest that gamma-glutamyltranspeptidase binding to vesicles is due to a papain sensitive sequence of amino acids and that the enzyme.vesicle complex closely approximates the interaction and orientation of gamma-glutamyltranspeptidase with brush border membranes.


Regulation of mitochondrial pyruvate carboxylation and gluconeogenesis in rat hepatocytes via  an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. Experiments were performed to determine if catecholamines can regulate control points in the gluconeogenic pathway, such as mitochondrial pyruvate carboxylation and pyruvate kinase activity, via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. Of a number of alpha agonists tested, only norepinephrine, epinephrine, and phenylephrine caused an increase in mitochondrial pyruvate metabolism. The effects of catecholamines on pyruvate carboxylation were not attenuated by 1-propranolol which abolishes changes in cyclic nucleotide levels but were blocked by alpha antagonists such as ergotamine, phenoxybenzamine, and phentolamine. Time course experiments demonstrated that the effects of catecholamines on the mitochondria and on carbohydrate metabolism correlated temporally with the concentration of epinephrine in the medium but not with the small changes in adenosine 3':5'-monophosphate. The effects of catecholamines appeared to require extracellular Ca2+ ion. The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation. Rather, catecholamines caused a smaller inhibition of pyruvate kinase activity than did glucagon. The effects of catecholamines on pyruvate kinase also appeared to be mediated by an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.


Regulation of rat hepatic stearoyl coenzyme A desaturase. The roles of insulin and carbohydrate. The rat hepatic stearoyl-CoA desaturation decreased by 3.7-fold in streptozotocin-induced diabetes. Insulin treatment of diabetic rats increased the enzyme activity by 7-fold. In marked contrast to glucose administration, fructose feeding in diabetic rats resulted in 20-fold stimulation of stearoyl-CoA desaturation, although both carbohydrates stimulated stearoyl-CoA desaturation in normal rats. Measurement of the microsomal electron transfer components showed no significant changes in the NADH-cytochrome b5 reductase activity or in the concentration of cytochrome b5. However, the activity of the terminal desaturase changed in a parallel fashion as the amount of terminal desaturase reflect changes in the overall desaturation. Supplementation of various microsomes with the saturating amount of purified terminal desaturase resulted in the formation of similar amounts of catalytically active complex and increased the stearoyl-CoA desaturation to the same level suggesting that the changes in the amount of terminal desaturase reflect changes in the overall desaturation. The results support the suggestion that both insulin and the intermediates of carbohydrate metabolism are involved in the regulation of terminal desaturase.


Stimulation by epinephrine of in vivo phosphorylation and inactivation of acetyl coenzyme A carboxylase of rat epididymal adipose tissue. Intraperitoneal injection of inorganic 32P into rats results in the incorporation of 32P into acetyl-CoA carboxylase without inactivation of the enzyme. Administration of epinephrine stimulates 32P incorporation and results in enzyme inactivation. Incubation of epididymal fat tissues with inorganic 32P also results in incorporation of 32P into carboxylase. This 32P incorporation reaches a maximum level in 3 h and it has no effect on carboxylase activity. Administration of epinephrine at the time of maximum phosphorylation (3 h) results in further phosphorylation and inactivation of carboxylase. Propranolol, a beta-adrenergic blocking agent which inhibits epinephrine action, blocks both the epinephrine-stimulated phosphorylation and the inactivation of the carboxylase. However, propranolol has no effect on that component of the phosphorylation which is unrelated to enzyme inactivation. These results establish that phosphorylation of carboxylase occurs in vivo at two different sites, only one of which results in enzyme inactivation. The phosphorylation site associated with enzyme inactivation is hormonally controlled.


Evidence for a high molecular weight factor(s) in serum which increases alkaline phosphatase specific activity in HeLa. HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC). For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum. The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%. The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.


Purification and properties of human erythrocyte inosine triphosphate pyrophosphohydrolase. Inosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Km of the enzyme is 1.3 X 10(-4), the Vmax = 1.2 X 10(-9) and the Keq = 3.8 X 10(4). Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ ions and by p-hydroxymercuribenzoate.


Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography-fluorescence detection method. A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These quanidino compounds are separated on a 6 x 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.


Quantitative analysis of amineptine (S-1694) in biological samples by gas chromatography--mass fragmentography. A sensitive, specific method for the quantitative analysis of amineptine in rat blood and brain is described. After extraction and purification amineptine is detected by mass fragmentography, monitoring the fragment ion at m/e 192. The method allows the quantitative analysis of as little as 50 ng of amineptine per ml blood and 200 ng per g for brain.


Analysis of cysteinyldopas, dopa, dopamine, noradrenaline and adrenaline in serum and urine using high-performance liquid chromatography and electrochemical detection. The catecholic amino acids, dopa, 2-S- and 5-S-cysteinyldopa, and 2,5-S,S-dicysteinyldopa were determined qualitatively in serum from patients with malignant melanoma by reversed-phase high-performance liquid chromatography, using electrochemical detection. In urine the catecholamines dopamine, noradrenaline and adrenaline were also determined qualitatively, as well as the above-mentioned compounds, in a single chromatographic run. The conditions were optimized by changing the pH of the mobile phase and by the addition of methanesulphonic acid. A comparison was made between the performance of four commercial reversed-phase packing materials containing chemically bonded octadecyl groups, using a standard mixture of catecholic amino acids. The influence of ionic strength, pH and amount of methanesulphonic acid on retention was investigated.


Gas-liquid chromatography of submicrogram amounts of drugs. V. Preparation of low-activity packed columns and their application to the toxicological analysis of underivatized polar drugs in the low nanogram range. When diatomaceous earth and glass columns are acylated prior to coating with a silicone liquid phase, then subjected to heat treatment in an atmosphere of nitrogen, gas chromatographic columns can be prepared that show a marked reduction in adsorption. These columns can be used with a nitrogen-specific detector to chromatograph unmodified polar compounds such as morphine and cyclobarbital in nanogram amounts. Virtually no alteration of peak shape and no variation of retention time are observed over the range 10(-6)-10(-9) g of polar drugs. This represents, for these polar drugs, an "improvement" in chromatographic capability of the order of about 1000-fold in comparison with the best conventional commercial columns. Application to toxicological analysis of morphine in urine is described.


The metabolism of floctafenin in man and rodents. Floctafenin (FFn), 2,3-dihydroxypropyl--N--(8--trifluoromethyl--4--quinolyl) anthranilate, a new nonnarcotic analgesic drug, was studied in man, mice, and the isolated perfused rat liver. In all species the drug is rapidly hydrolyzed to floctafenic acid (FFa). In seven volunteer subjects who each received a single oral dose of 400 mg floctafenin on an empty stomach, the blood concentration of FFa usually reached a maximum between 1 and 2 hours (mean 1.57 +/- 1.28 microgram/ml at 1.5 hours) and declined over the next 6 hours. Eight hours after drug administration the mean concentration of FFa in the blood of the volunteers was 0.1 +/- .05 microgram/ml. Approximately 25 per cent of the administered dose of floctafenin was recovered as FFa and hydroxy-FFa in the urine collected from each subject for 48 hours after drug administration. In mice each having received a single intraperitoneal dose of floctafenin (2 mg), the concentration of floctafenin declined by about 50 per cent in 15 minutes, and this decline was accompanied by a rise in the concentration of FFa that remained constant for 3 hours. The analgesic effect observed after administration of floctafenin to humans is likely to be mediated by its major metabolite, FFa. In these volunteers no free floctafenin was detected in the blood.


Relative systemic availability of sulfapyridine from commercial enteric-coated and uncoated sulfasalazine tablets. The absorption of sulfapyridine after a single 2.0-Gm oral dose of sulfasalazine, the drug of choice in the treatment of inflammatory bowel disease, as commercial uncoated and enteric-coated and uncoated tablets was evaluated in four healthy male adults. The peak plasma concentration of sulfapyridine after the enteric-coated tablets occurred at 20 hours on the average (compared to 14 hours for the uncoated tablets) and was only 50% of that attained from the uncoated tablets (P less than 0.05). The low relative extent of systemic availability of sulfapyridine from the enteric-coated tablets (65.5 +/- 6.3 per cent, mean +/- S.E.) compared to uncoated tablets may be due to absorption rate-dependent presystemic metabolism, since the relative extent of sulfapyridine absorption was 92.7 +/- 6.2 per cent compared to uncoated tablets. These findings suggest that enteric-coated and uncoated tablets of sulfasalazine are not bioequivalent. It remains to be determined whether the clinical efficacy of sulfasalazine from enteric-coated tablets is affected.


The effect of tablets composed of various mixtures of sugar alcohols and sugars upon plaque pH in children. Changes in plaque pH were examined following ingestion of tablets composed of of sorbitol (S) or xylitol (X) combined with various levels of dextrose (D), fructose (F), or sucrose (U). Pure S and X tablets caused a slight pH rise. The 3:1 ratios of S:D,, S:F, X:D, and X:F caused slight lowerings to plaque pH means of 6.3, 6.8, 6.7, and 6.7, respectively, while 3:1 S:U caused the pH to drop to 5.6. All the 1:1, 1:3 and 0;1 ratios caused large drops in plaque pH.


Biological apatite crystal disolution.

Biological apatite crystal disolution. The dissolution curves of human acid-treated enamel powder are characterized by a rapid initial step followed, after 10 or 15 minutes, by a second stage, with release of very small amounts of calcium. Increase in pH and addition of fluoride ions tend to diminish, for short intervals, the amount of dissolved apatites. For higher pH values the decrease is noted over a longer time. Study of the relative ionic variation of human enamel powder from young and adult patients subjected to acid requires infra-red vibration band analyses. Special attention was given to the absorptions at 610 cm-1 (phosphate groups), 880 cm-1 (CO32- in OH- sites and HPO42-), 1410 cm-1 (CO32- in phosphate sites) and 1550 cm-1 (CO32- in OH- position). All results were related to vibration band at 610 cm-1. A preferential loss of carbonates in the two possible sites was always observed for 10 or 15 minutes, followed by a high release of phosphate. The increase of pH or small amounts of fluoride displaced the preferential carbonate loss to longer times. In the presence of higher fluoride levels a disappearance of the preferential loss of carbonates was noted. A continuous increase of HPO42- in the absence of fluoride occurred; however a straight line with a smaller shape was present, with fluoride addition.


The effect of therapist presence on a desensitization program for test anxiety. This study investigated the effect of varying degrees of therapist presence on outcome measures of a test anxiety program on systematic desensitization. Three treatments, which included face-to-face administration by a therapist, some therapist involvement, and fully automated administration were compared to a waiting list control group. It was found that all three treatments were effective when compared to the control group in terms of reduction of reported anxiety as measured by the STABS. It was noted that clients who used the automated program were more likely to terminate prematurely. It was concluded that, while all of the treatments may be effective in a laboratory setting, the presence of a counselor would seem to be important for the use of this type of program in a service agency.


Survival and growth of non-cholera vibrios in various foods. A study was made of the growth of three strains of non-cholera vibrio in a range of foodstuffs and of the effect of temperatures and pH on their ability to grow. Growth was tested at 4 degrees, 10 degrees, 22 degrees, 30 degrees, 37 degrees and 43 degrees C in a range of foods likely to be incorporated into cold hors d'oeuvres, e.g. egg, cream, rice, cold meat, seafood, aspic and mayonnaise. Non-cholera vibrios grew well in all these foods except mayonnaise, the rate of growth increasing with increased temperature of storage. At acid pH values the organisms died or grew very poorly but growth improved as the pH became more alkaline. None of the three strains showed any resistance to heat, an initial inoculum of greater than 10(7) organisms/g was reduced to less than 100 organisms/g in 2--3 min at 55 degrees C.


Cell-mediated suppression of the fifth component of complement in mice. Suppression of levels of circulating C5 in (C5- C5+)F1 hybrids by administration of (C5- C5-) parental lymphoid cells in the neonatal period has been accomplished with the three strain combinations tested ((SWR X RIII)F1, (A/He x RIII)F1, and (SWR X DBA/1)F1). Suppression was shown to be specific for C5 and not accompanied by reductions of C1, C2, C6, or other major groups of blood proteins. This demonstrated that the C5 reduction was not due to activation of complement (C) with resultant hypercatabolism of C components. When there was a concurrent chronic GVH reaction induced by lymphoid cells administered to offspring of H-2 incompatible parents, there was usually a resultant hypergammaglobulinemia that was also unrelated to the presence or absence of C5 suppression. Effective suppression required preimmunization of either the cell donor, the mother of the F1 hybrids, or both. This suggests that either two cell types or a single cell plus a humoral factor are required for suppression in this system.


In vivo activity of amoxicillin and ampicillin against gram-positive bacteria: results of prophylactic studies. In prophylactic treatment of intraperitoneal infections with Streptococcus pneumoniae (types 1 and 2) and Streptococcus pyogenes in mice, amoxicillin had a definite advantage over ampicillin in terms of protective effect. When the infecting agent was given to mice so as to produce an infection in the brain or lung (for example, S. pneumoniae given intracranially or intranasally), amoxicillin was also more effective prophylactically than ampicillin.


The productivity potential of physicians' assistants: an integrated approach to analysis. Although many previous analytical studies indicate that physicians' assistants can substantially increase productivity and reduce costs, the utilization of physicians' assistants in ambulatory health care settings has grown at a disappointing rate. This apparent discrepancy may be explained in part through close examination of the models used in the analytical studies. This paper describes the limitations of previous studies and shows how analysis can be improved through the use of a combined optimization-simulation model. The model is applied to a hypothetical example to demonstrate how productivity and income benefits can be overstated, and to test the sensitivity of such benefits to a range of management policies for the ambulatory care practice regarding patient load, physical resources, appointment scheduling, and more flexible assignment of providers to patients.


The role of H-2 and Ia antigens in graft-versus-host reactions (GVHR). Presence of host alloantigens on donor cells after GVHR and suppression of GVHR with an anti-Ia antiserum against hose Ia antigens. By using an indirect immunofluorescence technique, the presence of host cell derived H-2K, H-2D, and Ia alloantigens on donor cells recovered from recipient spleens after a graft-versus-host response (GVHR) was demonstrated. Mapping studies indicated that only host K, D, and I-A region gene products could be identified on the donor cells. Host I-E/C- and I-J-subregion products were not absorbed by donor cells. Treatment of activated donor cells with anti-Ly sera plus C' revealed that donor cells carrying host Ia antigens have a Ly-1+,2-,3- phenotype, whereas donor cells carrying H-2K and H-2D host antigens have a Ly-1-,2+,3+ phenotype. A GVHR that resulted from only an I-region incompatibility was suppressed by the injection of recipient mice with an anti-Ia antiserum directed against self Ia antigens. The degree of suppression was proportional to the amount of anti-Ia antiserum administered.


Immunological studies of T-cell receptors. II. Limited polymorphism of idiotypic determinants on T-cell receptors specific for major histocompatibility complex alloantigens. These studies explore the extent of genetic polymorphism in the expression of anti-MHC receptors by T cells in different strains of rats. This question was approached with the use of the model of specifically induced GVH resistance in F1 rats which has been shown to reflect a specific T-cell mediated immune response against parental strain T cell anti-MHC receptors specific for host alloantigens. When A/B F1 rats derived from MHC incompatibile matings are immunized with lymphocytes from one parental strain (A they display a specific resistance to anti-B GVH reactivity caused by T cells from that parental strain, but not anti-AGVH reactions from the other. In addition, they resist anti-B GHV reactivity by T cells from third-party donors (C, D, E,...), a finding taken to indicate that the idiotypes of anti-MHC receptors on T cells, recognized by other T cells, show little or no polymorphism. This conclusion suggests that anti-MHC receptors are shared in the species and may be encoded, at least partially, by germ-line genes.


Isolation of type-specific polysaccharide antigen from group B type Ib streptococci. Group B streptococcus type Ib (strain H36B) was subjected to digestion with extracellular muralytic enzymes prepared from Streptomyces albus. Type Ib-specific polysaccharide antigen was isolated from the lysate by alcohol precipitation and Sepharose 6B chromatography. The purified type Ib antigen has a Kd value of 0.31 on a Sepharose 4B column and contains four sugars, galactose, glucose, N-acetyl glucosamine, and sialic acid in a molar ratio of 2.05:0.86:1.00:0.90. Acid treatment (pH 2.0) of this polysaccharide results in partial degradation of the antigen (Kd = 0.41 on Sepharose 4B) with the loss of 93% of the sialic acid. The molar ratio of the remaining sugars in the polysaccharide remains identical to that in the native one. This suggests that the sialic acid is at the terminal position in the molecule. Both intact and acid-treated antigen cross-react with some type Ia and type Ic antisera as a result of the common Iabc determinant, but not with type II and type III antisera. Absorption studies indicate that Ib-specific determinant and Iabc determinant are on the same molecule and that sialic acid is not the cross-reactive determinant.


[Horseradish peroxidase: a study of the kinetics and the determination of optimal reaction conditions, using hydrogen peroxide and 2,2'-azinobis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as substrates (author's transl)]. The reaction of the two substrates hydrogen peroxide and ABTS with horseradish peroxidase was studied kinetically. Enzyme activity was determined as a function of substrate concentration and pH. Michaelis constants were determined for the two substrates at various pH values. It was found that the affinity of the enzyme for ABTS decreases with increasing pH, and that with higher ABTS concentrations the pH optima of the reaction are shifted towards neutrality. Maximal rate is reached at pH 4.2 with an ABTS concentration of 2 mmol/l. For hydrogen peroxide the data show that the dissociated O2H- is the proper substrate, its affinity for the enzyme being independent of pH. The two substrates show competitive binding to the peroxidase, and each therefore influences the binding constant of the other. A procedure is proposed which allows the determination of peroxidase down to a concentration of 10 ng/l or 2.5 x 10(-13) mol/l.


Age factors in patient-therapist relationship. This paper reports on the effects of therapist and patient age as factors in the evaluation and treatment of adult psychiatric outpatients. Therapist ratings and patient self-ratings are contrasted in three age groups. Rating scales include symptomatology, motivation, insight, and prognosis. In addition, a chart review follow-up of 68 patients includes disposition and drop-out. The data suggest that older patients are perceived as sicker, but less treatable than younger patients or patients of the same age group as the therapist. Residents express a strong preference for treating younger patients, but more readily develop a treatment relationship with same age patients. Both older and younger patients were significantly less likely to remain in treatment. Age of therapist and patient as a significant, frequent unrecognized factor in psychotherapeutic intervention is discussed and modifications in psychiatric training programs are suggested.


Lack of complaints in schizophrenics with tardive dyskinesia. Eighteen of a population of 518 outpatients were found to fulfill diagnostic criteria for tardive dyskinesia. None of these patients complained to their therapists of their symptoms, and eight of them were not even aware of them. Five of the unaware patients were actively delusional or hallucinating. Since all eighteen were chronic schizophrenics on neuroleptics, these findings suggest that this group may not report their symptoms. The findings are important because early diagnosis of tardive dyskinesia allows more chance for intervention with satisfactory results.