Regulation of placental enzymes of the carbohydrate and lipid metabolic pathways. The activity of enzymes with a regulatory function in the pathways of glycolysis, gluconeogenesis, NADPH generation and fatty acid synthesis was measured in the placenta and liver of rats. Compared with the liver, a high activity of pyruvate kinase was found in the placenta, indicating a high glycolytic potential; a small capacity for gluconeogenesis was also present and a moderate to low activity of enzymes associated with lipogenesis. The activity of all placental enzymes fell from day 15 to 20 of gestation irrespective of the pathway they represented. The pattern of decline continued when the gestation was prolonged up to day 26 by the administration of chorionic gonadotropin. The rates of activity disappearance over 11 days of gestation differed for each enzyme, with half-lives ranging from 2.7 days for NADP-malate dehydrogenase to 7 days for glucose-6-phosphate dehydrogenase. In contrast, the activity of hepatic enzymes either remained unchanged or showed individual adaptation to the advancing pregnancy. The regression in placental metabolic capacity after day 15 of gestation was also evident by the decrease in glucose uptake and its channelling to lactate, CO2, glycerol and fatty acids. In addition, placental ageing was associated with triglyceride accumulation, mainly due to the decrease in free fatty acid oxidation. Treatment of pregnant rats with several hormones, while markedly affecting the hepatic enzyme activities, failed to induce appreciable changes in the corresponding placental enzymes. This was illustrated in the case of triiodothyronine treatment. Similarly, insulin deficiency induced by streptozotocin failed to elicit adaptive changes in placental enzyme activities typical of diabetes like those occurring in the maternal liver; some converse responses in the placenta were attributed to hyperglycaemia. On the other hand, responses in some fetal liver enzymes were suggestive of fetal hyperinsulinaemia. These observations indicate that placental enzymes are not susceptible to endocrine regulation and imply that placental metabolism is largely independent of the physiopathological alterations affecting the maternal organism. The gradual activity decreases with gestation suggest that the enzyme complement of the placenta, once developed, is designed to last through its limited lifespan without continuous replenishment. Within this context, no mechanism seems to operate to ind1ce the adaptive synthesis of individual enzymes, and the age of the placenta appears to be the primary factor determining its enzyme activity and metabolic performance.


Pathogenesis of duodenal ulceration produced by cysteamine or propionitrile: influence of vagotomy, sympathectomy, histamine depletion, H-2 receptor antagonists and hormones. Insight into the pathogenesis and etiology of experimental duodenal ulceration was sought by studying the modulation of this disease in rats by selective vagotomy, chemical sympathectomy, histamine depletion, histamine H-2 receptor antagonists (eg, metiamide, cimetidine), or endocrine ablations. Gastric secretion was examined in intact and pylorus-ligated animals. The formation of duodenal ulcers induced by the administration of propionitrile or cysteamine was abolished by vagotomy, decreased by sympathectomy, histamine depletion, histamine H-2 receptor antagonists, hypophysectomy, thyroidectomy, or adrenalectomy. Cimetidine and metiamide exerted a dose-dependent antiulcer effect, but metiamide enhanced the mortality of rats given propionitrile or cysteamine. The non-ulcerogen derivative of cysteamine, ethanolamine, did not increase mortality when given in combination with metiamide. The gastric hyperacidity elicited by cysteamine was reduced by metiamide or vagotomy, the latter being more effective in this respect. Thus, the chemically induced duodenal ulcer in rats resembles the human peptic ulcer disease in sensitivity to therapeutic modalities and may serve as an appropriate model to study the role of neural, hormonal, and other factors in the etiology and pathogenesis of this disorder.


[Parasympathicolytic drugs in treatment of obstructive lung diseases (author's transl)]. In order to investigate the effect of ipratropium bromide in antigen induced bronchial obstruction 8 series of investigations were performed in patients with extrinsic asthma using bronchial provocation tests. The broncholytic effect of ipratropium bromide (0.1 and 0.25 mg in 1 ml solution) was significantly lower when compared with fenoterol and salbutamol and not clearly distinct from untreated controls, when airway resistance, thoracic gas volume and specific airway resistance were measured. No correlation of the effect of ipratropium bromide with bronchial reactivity to histamine, basal values and antigen induced obstruction as well as corticosteroid dependency could be ascertained. Increase of dosage did not lead to improved effectiveness. In contrast protective application of ipratropium bromide before antigen inhalation led to prevention of obstructions did disodium cromoglycate. It is concluded that regular prophylactic application of ipratropium bromide is more effective than bronchospasmolytic application. All forms of obstructive pulmonary disease are thus an indication for parasympathicolytic drugs provided they are used regularly.


Cefuroxime: a review of its antibacterial activity, pharmacological properties and therapeutic use. Cefuroxime is a new semisynthetic cephalosporin for parenteral administration. It is resistant to destruction by beta-lactamases produced by staphylococci and most Gram-negative aerobic bacteria and is active against many bacteria resistant to cephalothin. Cefuroxime is the most active of the cephalosporins against gonococci and Haemophilus influenzae particularly against beta-lactamase producing strains. Given by intramuscular or intravenous injection cefuroxime is effective against a wide variety of infections caused by Gram-positive or Gram-negative aerobes, but has no effect against infections caused by Pseudomonas aeruginosa or B. fragilis. Cefuroxime is of value in the treatment of respiratory infections due to Haemophilus influenzae and Streptocococcus pneumoniae and is useful against cephalosporin-resistant Klebsiella and Enterobacter infections. Cefuroxime is an alternative to spectinomycin for the treatment of beta-lactamase producing Neisseria gonorrhoeae infections. It is generally well tolerated and appears not to be nephrotoxic when given alone at usual dosages.


Bromocriptine in the treatment of parkinsonism. Bromocriptine alters the motor behaviour of animals and improves the motor defect of parkinsonism. Changes in movement are accompanied by biochemical changes in the central nervous system, consistent with the idea that bromocriptine has a dopamine agonist action in the basal ganglia and also in the mesolimbic system and hypothalamus. The overall anti-parkinsonian effect of bromocriptine is similar to that of l-dopa alone or with benserazide (a decarboxylase inhibitor) when optimum doses are used, although individual patients may respond better to 1 drug than to the other.


Drugs, alcohol and driving.

Drugs, alcohol and driving. Driving a car is a complex psychomotor and perceptual task which is subject to impairment by many factors. Several workers have studied the potential effects of drugs and alchol in crash production by epidemiological and laboratory studies. Both types of studies have yielded useful data but their limitations must be borne in mind when applying the results in pratice. Alcohol is obviously the most common single cause of traffic accidents. A progessively increased risk with increasing blood alcohol levels is well documented; fatigue and/or drugs increase this risk. Drugs are related much more infrequently to traffic accidents although on the basis of statistics, there is a potential risk with drug use. However, drugs alone are not as important as alcohol. The most significant drugs as regards driving risk are obviously certain antianxiety agents, hypnotics, stimulants, hallucinogens, marihuana, lithium and narcotic analgesics, as well as ganglionic blocking agents, insulin and sulphonylurea derivates. Patients should not drive after taking these drug until they are objectively fully alert and capable. Anticholinergics, antihistamines, antidepressants, antipsychotics, phenybutazone, indomethacin, alpha-methyldopa, and beta-blockers may in some cases cause central side effects (e.g. drowsiness) strong enough to affect driving performance. After starting therapy with these drugs, or after a significant change in dose, driving should be avoided until it is known that unwanted effects do not occur. Psychotropic drugs may enhance the deleterious effect of alcohol, and with most hypnotics there is still an effect the next morning. Some drugs (e.g. anticonvulsants or antiparkinsonian drugs) may make driving safer, but the disease (epilepsy, Parkinsonism, cardiovascular diseases, psychic disorders, etc.) ofter precludes driving. Clinicians should warn their patients about an impairment of driving skills if this is likely to occur due to the drug or the illness concerned.


Differential selectivity of several barbiturates on experimental seizures and neurotoxicity in the mouse. Six barbiturates with diverse time-action characteristics--thiopental, pentobarbital, butabarbital, phenobarbital, diphenylbarbiturate, and barbital--were evaluated for "anticonvulsant" and "neurotoxic" effects. For the former, the MES test, clonic seizures induced by pentylenetetrazol, 90 mg/kg, s.c., and maximal seizures produced by pentylenetetrazol, 200 mg/kg, s.c., were employed. For the latter, we used a rotorod technique. Time to peak activity in the MES test was employed as the time for other tests. Pentobarbital required at least neurotoxic doses to produce substantial "anticonvulsant" activity, its protective index ranging from 0.79 to 0.98 in the three tests. Among the drugs tested, phenobarbital and diphenylbarbiturate exhibited the most favorable protective indices, ranging from 2.71 to 3.41 for phenobarbital and from 3.85 to 5.0 for diphenylbarbiturate. Barbital, another drug with a prolonged duration of action, exhibited a range from 0.84 to 2.81. Although a prolonged duration of action is an important characteristic for antiepileptic activity, this property does not confer per se a favorable protective index.


Dual localization of long-chain acyl-CoA hydrolase in rat liver: one in the microsomes and one in the mitochondrial matrix. Subcellular fractionation studies of rat liver localized the activity of palmitoyl-L-carnitine hydrolase to the microsomal fraction whereas palmitoyl-CoA hydrolase activity was found both in the microsomal fraction and in mitochrondria. An unusual biphasic sataration curve for palmitoyl-CoA was observed when intact mitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl-CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin fractionation of rat liver mitochondria demonstrated that a palmitoyl-CoA hydrolase was associated with the matrix fraction. Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl-CoA were higher for the microsomal enzyme than for the matrix-associated enzyme.


The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia. Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.


Light-induced proton transport by chloroplasts suspended in fluid media at sub-zero temperatures: kinetics and stoichiometry. 1. Chloroplasts suspended in a medium containing ethanediol and water (1 : 1, v/v) at -16 degrees C show light-induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics. 2. A 1 ms flash caused a single turnover of the photochemical centres at -16 degrees C. Under the same conditions 3H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor. 3. The flash-induced proton uptake was exponential and monophasic with t1/2 = 3 s. The flash-induced proton release into the thylakoid interior was biphasic, with half-times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water. 4. The half-time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half-time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.


Kinetic studies of the interaction between MS2 phage and F pilus of Escherichia coli. The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation (kd) of the MS2-phage--F-pilus complex is very slow and follows first-order kinetics with a half-life of 4.2 h at 30 degrees C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half-life of the complex is about 1.0 min. The rate of association is very fast and follows second-order kinetics with the rate constant for association (ka) being 8 x 10(7) M-1 s-1 at 30 degrees C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non-electrostatic respectively. The mechanism of the binding reaction is discussed.


1H nuclear-magnetic-resonance studies of porcine lutropin and its alpha and beta subunits. The titration curves of the histidine residues of porcine lutropin and its isolated alpha and beta subunits have been determined by following the pH-dependence of the imidazole C-2 proton resonances. The isolated alpha subunit contains a buried histidine, whose C-2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2). The four histidine C-2 proton resonances have been assigned to specific residues in the amino-acid sequence, by means of deuterium and tritium exchange experiments on the alpha subunit and its des(92-96) derivative. The histidine with a pK of 3.3 is identified as His-alpha87. The effects of pH on tyrosine and methyl proton resonances show that the titration of His-87 in the isolated alpha subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition. The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.


Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite. 1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.


An acid protease in human erythrocytes and its localization in the inner membrane. The isolation of erythrocytes of high purity from human blood was achieved by a combination of the two well established methods cells in erythrocyte preparations of different purities was studied. The acid protease activity was recovered to a level comparable with the recovery of erythrocytes, while the neutral protease activity as detected by the release of acid-soluble peptides from hemoglobin or casein disappeared in proportion to the removal of white blood cells. An acid protease was solubilized from the membranes of the purified erythrocytes by the extraction with 1-butanol. The enzyme was active in a pH range from 2 to 4, and sensitive to pepstatin. It was  named pH-3 protease after its pH optimum. Sealed ghosts with right-side-out membranes and inside-out vesicles with reverted membranes were prepared from the purified erythrocytes and compared with respect to pH-3 protease activity for its latency as well as its inactivation by tryptic digestion. The results obtained indicate that pH-3 protase is localized on the inner surface of erythrocyte membranes. The self-digestion experiments at pH 4 using the sealed ghosts showed higher availability to pH-3 protease of spectrin and IVa protein than the other membrane proteins, also suggesting the localization of an acid protease in the inner membranes of erythrocytes.


Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart. A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.


Fabry's disease with familial lymphedema of the lower limbs. Case report and family study. The case of a 49-year-old man with Fabry's disease (FD), confirmed by histopathological findings of kidney and skin biopsies and enzymatic studies, is reported. Clinical symptoms mainly consisted in severe neurological involvement, and in conspicuous lymphedema of the lower limbs. Two decreased brothers of the patient were also affected with symptons strongly suggesting FD, as well as the lymphedema of the lower limbs. On the basis of these data, the association of FD with familial lymphedema of the lower limbs is discussed: a lipid accumulation in the lymphatic as well as the blood vessel wall is proposed as a possible explanation; the hypothesis of an inborn error in the development of the lymphatic system, controlled by a gene closedly associated with the FD gene on the same chromosome can also be advanced.


Transfer in vitro of three benzodiazepines across the human placenta. A comparative study of the placental transfer to the foetus of three benzodiazepines was performed using a dual perfusion system of the human placental lobule. A transport fraction was calculated for each benzodiazepine and was compared with reference substances. Relative to antipyrine, the transport fraction of diazepam was 85%, and that of nordiazepam was 84%. The transport fraction of clorazepate represented only 20% of that of tritiated water. The relatively high transfer of diazepam and nordiazepam can be attributed to their high lipid solubility, and the lower transfer of clorazepate is due to its polar nature. It is suggested that in certain instances this benzodiazepine may be of especial value to obstetricians.


Pharmacokinetics of clorazepate in pregnant and non-pregnant women. A single dose of clorazepate 20 mg was injected i.m. in 7 pregnant and 7 non-pregnant women. Blood samples were collected for one week, and urine was collected for 24 h after the dose. The concentrations of clorazepate and its metabolite nordiazepam were determined by electron capture gas liquid chromatography. There was no difference between the two groups on physical examinations. Clorazepate was rapidly absorbed and the peak concentration was reached within 2 h. Mean pharmacokinetic parameters for clorazepate were absorption half life 0.77 h in pregnant women and 0.56 h in non-pregnant women; elimination half life 1.3 h in pregnant women and 2.0 h in non-pregnant women; volume of distribution: 0.43 1 . kg-1 in the pregnant women and 0.33 1 . kg-1 in non-pregnant women. Nordiazepam reached its peak concentration within 12 h after dosing; its mean half life of elimination was 180 h in pregnant women and 60 h in non-pregnant women. Within 24 h, 1.3% of the clorazepate was recovered in urine from pregnant women and 7% in urine from the non-pregnant women.


Pharmacokinetics of the placental transfer and distribution of clorazepate and its metabolite nordiazepam in the feto-placental unit and in the neonate. Clorazepate 20 mg was given i.m. to 49 mothers during the first stage of labour. The elimination of the drug was studied in 27 newborns produced by these mothers. The same dose was given to 13 women who underwent amniocentesis and to 7 women who were breast-feeding. "Total nordiazepam", i.e. the sum of clorazepate and its metabolite nordiazepam, was determined by gas-liquid chromatography in maternal blood, umbilical cord blood (both arterial and venous), amniotic fluid and in milk. Clorazepate was found to cross the placental barrier slowly, but nordiazepam was transferred more rapidly. Nordiazepam was found in the milk and in the blood of neonates after breast-feeding had started.


The effect of rifampicin on norethisterone pharmacokinetics. The pharmacokinetics of norethisterone have been studied in 8 women during and one month after treatment with rifampicin (450--600 mg/day). Rifampicin caused a significant reduction in the A.U.C. of a single dose of 1 mg norethisterone from 37.8 +/- 13.1 to 21.9 +/- 5.9 ng/ml X h (p less than 0.01). The plasma norethisterone half life (beta-phase) was also reduced from 6.2 +/- 1.7 to 3.2 +/- 1.0 h (p less than 0.0025). In one additional woman on long term oral contraceptive therapy the 12 hour plasma norethisterone concentration was reduced by rifampicin from 12.3 ng/ml to 2.3 ng/ml. Rifampicin caused a significant increase in antipyrine clearance, 6 beta-hydroxycortisol excretion and plasma gamma-glutamyltranspeptidase activity but there was no significant correlations between changes in these indices of liver microsomal enzyme induction. There was a significant correlation between the percentage increase in antipyrine clearance and the percentage decrease in norethisterone A.U.C. during rifampicin. The changes in norethisterone pharmacokinetics during rifampicin therapy are compatible with the known enzyme inducing effect of rifampicin.


Abrupt withdrawal of beta-blocking agents in patients with arterial hypertension. Effect on blood pressure, heart rate and plasma catecholamines and prolactin. Chronic treatment with beta-blockers was interrupted abruptly in six patients with arterial hypertension. Three patients, who had experienced symptoms during a previous withdrawal, again complained of transient palpitations, tremor, sweating, headache and general malaise. A significant increase in standing blood pressure (BP) and heart rate (HR) was noted after 24 h. The standing HR reached a maximum after 48 h and had decreased significantly on the 7th day (p less than 0.005). There was a strong tendency to greater increase in standing BP and HR in the patients who experienced symptoms than in those who did not. Plasma concentrations of noradrenaline, adrenaline and prolactin did not change significantly. Thus, beta-blocker withdrawal symptoms are reproducible and are indicative of a transient sympathetic hyperresponse. The increased activity is not likely to be caused by increased production of circulating catecholamines, but rather by increased sensitivity of the beta-receptor.


beta-Adrenoceptor blocking effects and pharmacokinetics of pindolol. A study in hypertensive Africans. The beta-adrenoceptor blocking effects of pindolol were compared with those of a placebo in a double-blind trial in twelve hypertensive Africans. Heart rate and arterial blood pressure were measured at rest and immediately after exercise, before and at intervals up to 8 h after oral administration of the drugs. Plasma levels of pindolol were also determined. Pindolol reduced systolic blood pressure and antagonised exercised-induced tachycardia. The mean time to peak level of pindolol was 1.9 h and the mean half-life was 4.2 h. Comparison of plasma levels of pindolol and beta-adrenoceptor blocking activity showed good correlation between them. It is concluded that the pharmacokinetics and beta-blocking effects of pindolol in Africans are not dissimilar from published data for other races.


Effect of short-term high-dose treatment with methenamine hippurate on urinary infection in geriatric patients with an indwelling catheter. IV. Clinical evaluation. An evaluation has been made of the clinical and laboratory effects of short-term (34 days), high-dose (2g x 3 daily) treatment with methenamine hippurate (MH) of 14 geriatric patients with an indwelling catheter and clinical features of urinary tract infection. During MH treatment the number of catheter changes was halved, each catheter remaining in situ for an average of 12.0 days as compared to 6.2 days in the pre-treatment control period and 5.2 days in the post-treatment control period; the difference is significant (p = 0.008; Friedman two-way analysis of variance). Urine pH was reduced (pH 7.0--6.5--7.0; p = 0.01) and the standard bicarbonate in blood was slightly elevated (24.1--25.7--25.0 mmol/l; p= 0.008) during the MH treatment period, when compared to pre- and post-treatment control periods. It is suggested that MH treatment reduced the complications associated with indwelling catheters due to reduction in urine pH, bacteriuria, and pyuria. Blockage of catheters is thought to be due to intraluminal salt precipitations with trapping of clumps, and is primarily not correlated with urine viscosity.


Beta-adrenoceptor blockade by atenolol, metoprolol and propranolol in the anaesthetized cat. The inhibitory effects of atenolol, metoprolol and propranolol on isoprenaline-induced tachycardia, broncho-relaxation and vasodilatation were investigated in the reserpinized and anaesthetized cat. In low doses all three antagonists inhibited the heart rate response to isoprenaline, the order of potency being propranolol greater than metoprolol greater than atenolol. While propranolol inhibited the bronchodilation and vasodilation responses to isoprenaline in the same dose range as it blocked the heart rate response, atenolol and metoprolol had to be given in considerably higher doses to block these effects. The results indicate that both metoprolol and atenolol, in contrast to propranolol, are selective beta1-adrenoceptor antagonist. No statistically significant difference in the degree of selectivity was found between metoprolol and atenolol. The three compounds were devoid of intrinsic beta-mimetic activity.


An automated superfusion technique for measuring circular muscle contraction. Effects of serotonin on intra- and extracranial arteries. A new in vitro technique is described and its advantages are demonstrated: "true" circular contraction is measured; arteries are in cascade, permitting comparison of intra- and extracranial arteries from the same animal; the mechanical influences on contractility are reduced; long-lasting experiments of up to 50 h can be performed; the procedure is fully automated. The maximal effect of an agonist was not only markedly modified during the first 2--4 h after fixing the arteries in the apparatus but also slightly during the next 3--4 h. After the initial stabilization period, the arterial response to an agonist remained highly reproducible for 24--48 h (standard deviation not exceeding 7%). This was demonstrated in all the types of arteries tested. With serotonin as agonist, there was a significant difference in --log ED50 values for intracranial arteries (basilar 8.70, middle cerebral 8.72) vs. extracranial (lateral nasal 8.15; facial 8.14) and peripheral (branch of saphenous 8.14) arteries.


Binding characteristics of 3H-prazosin to rat brain alpha-adrenergic receptors. 3H-Prazosin binding to membranes from rat brain is saturable, Bmax, 77 fmol/mg protein, of high affinity, KD, 0.28 nM and with a drug specificity indicating that it labels alpha-adrenergic receptors. The ranking of alpha-antagonists for these binding sites suggests that 3H-prazosin may be useful in identifying a subpopulation of alpha-receptors (alpha1) in the central nervous system.


Embryology of the diffuse neuroendocrine system and its relationship to the common peptides. The diffuse neuroendocrine system is constituted by the cells, now more than 40 in number, of the central and peripheral divisions of the amine precursor uptake and decarboxylation (APUD) series. At one time presumed to be derived from a common "neural" ancestor, all are now deemed to be "neuroendocrine-programmed," arising either in the embryonic epiblast itself or in one of its principal descendants. The APUD cells produce more than 35 physiologically active peptides and a small number of equally active amines. Within the last 3 years, 17 of these peptides have been identified jointly in endocrine cells and in neuronal cell bodies or processes. Sharing in this way a neural and an endocrine location and site of production, they are called the "common peptides." The diffuse neuroendocrine system is to be regarded as a third division of the nervous system, whose products suppress, amplify, or modulate the activities of the other two divisions. The relationship of its products to the cells and processes of these two divisions is currently the object of intensive inquiry.


[Influence of the vertebral nerve on several indices of cerebral blood flow]. The dogs and rabbits, electrical stimulation of the vertebral nerve reduced the pressure in the carotid and the vertebral--basillar arterial systems leaving the general arterial pressure unaltered. Simultaneously the values of pH, PO2 PCO2, and buffer bases decreased in the blood flowing from the dog brain. The data obtained suggest that stimulation of the vertebral nerve reduces the blood flow in the vertebral artery which, in conditions of occlusion of the contralateral vertebral artery, leads to the circulatory regional hypoxia of the brain.


[Receptor substances of brain mitochondria sensitive to neuromediators]. The action of some neurotransmitters and their derivatives on succinate dehydrogenase and cytochrome oxidase of rat brain mitochondria was studied in vitro. Alpha--adrenoreceptor blocking agents phentolamine and dibenzyline abolished the inhibitory action of the native forms of catecholamines (norepinephrine, epinephrine, isoproterenol) on the enzymatic activity under study. Relationships among catecholamines, cyclic 3',5'--AMP, and cortisone in their influence on these enzymes were studied. The data obtained indicate the existence of alpha--type adrenoreceptors in the brain mitochondria which can be responsible for the regulatory influences of catecholamines on functional activity of mitochondria.


[Rat brain nuclease activity during learning with emotionally different reinforcement]. Acid and alkaline activity of nucleases of the rats trained with emotional positive or negative reinforcement was estimated in the neocortex, hippocampus, midbrain, and in caudal portions of the brain-stem, using native and denaturated DNA as a substrate. The results showed the total increase in nuclease activity during learning. Nevertheless the dynamics of enzyme activation was different depending on the emotional state of rats during learning. The most active enzyme was found in the caudal portion of the brain-stem.


A versatile new sustained-action neuroleptic: pipotiazine palmitate in psychiatric practice. The long-term clinical effects of pipotiazine palmitate were tested in 206 men and women who were either not responding well to their previous neuroleptic therapy or who were negligent about pursuing protracted oral drug therapy. Of the 206 patients, 130 were suffering from some form of chronic schizophrenia; the remainder presented with depression, psychoneurotic or behavioural disorders. Pipotiazine palmitate, a long-acting depot neuroleptic, was given as a monthly intramuscular injection for up to 23 months. The average starting dose was 50 mg/injection and the average final dose was 65 mg/injection. These doses were somewhat lower than those usually reported in the literature, however all but a few patients received oral neuroleptics or antidepressants concomitantly. Psychiatric testing using the Brief Psychiatric Rating Scale revealed that significant improvement was achieved over time in all diagnostic groups represented. Individual as well as cumulative scores improved steadily for 6 momths at which time symptomatology was minimal in most patients. Pipotiazine palmitate was well tolerated, and only seven (3.4%) of the 206 patients had to interrupt therapy because of unwanted effects. The most frequent side-effects were extrapyramidal symptoms, particularly tremor and rigidity, yet these effects led to the discontinuation of therapy in only five patients.


Plasma corticosterone response to serotonin altering drugs in the 3-day-old rat. Three-day-old rats were injected with various neurotransmitter altering agents to demonstrate a functional relationship between these drugs and plasma corticoid levels. Plasma corticosterone levels were increased after injection of methiothepin, methysergide, and 5-hydroxytryptophan (5-HTP), but were not changed by cholinergic, adrenergic, and dopaminergic compounds. Imipramine alone had no effects on plasma corticoids but in combination with 5-HTP resulted in a more sustained response than 5-HTP alone. Afunctional relationship between plasma corticosterone and serotonin receptors has been demonstrated in the 3-day-old rat. The presence of this relationship just after birth suggests the possibility that serotonin may be a mediator of early experience effects on later adrenocortical function.


Interaction between acute gastric ulcer and epinephrine-induced mucosal erosions in the rat: the significance of gastric acid secretion. Gastric mucosal erosions were produced in rats by pyloric occlusion and intraperitoneal injection of epinephrine. Gastric ulcer was induced by application of acetic acid to a limited area of the stomach. pH, total amount of acid and volume of gastric juice were determined 5 h after pyloric occlusion. The presence of an acute acetic acid ulcer partly inhibited the development of mucosal erosions induced by pyloric occlusion and epinephrine. The gastric ulcer was associated with decreased acid secretion. A positive correlation was obtained between the degree of mucosal erosions and the total amount of acid in the stomach.


The stimulus-secretion coupling of glucose-induced insulin release. XXXV. The links between metabolic and cationic events. When isolated rat islets were exposed to glucose, the concentrations of NADH and NADPH, and the NADH/NAD+ and NADPH/NADP+ ratios were increased. The dose-response curve resembled that characterising the glucose-induced secondary rise in 45Ca efflux, displaying a sigmoidal pattern with a half-maximal value at glucose 7.5 mmol/l. The glucose-induced increase in NAD(P)H was detectable within 1 min of exposure to the sugar. Except for the fall in ATP concentration and ATP/ADP ratio found at very low glucose concentrations (zero to 1.7 mmol/l) no effect of glucose (2.8-27.8 mmol/l) upon the steady-state concentration of adenine nucleotides was observed. However, a stepwise increase in glucose concentration provoked a dramatic and transient fall in the ATP concentration, followed by a sustained increase in both O2 consumption and oxidation of exogenous + endogenous nutrients. This may be essential to meet the energy requirements in the stimulated B-cell. Although no significant effect of glucose upon intracellular pH was detected by the 5,5-dimethyloxazolidine-2,4-dione method, the net release of H+ was markedly increased by glucose, with a hyperbolic dose-response curve (half-maximal response at glucose 2.9 mmol/l) similar to that characterising the glucose-induced initial fall in 45Ca efflux. It is proposed that the generation of both NAD(P)H and H+ participates in the coupling of glucose metabolism to distal events in the secretory sequence, especially the ionophoretic process of Ca2+ inward and outward transport, and that changes in these parameters occur in concert with an increased turn-over rate of high-energy phosphate intermediates.


Electron microscopic radioautographic identification of the ECL cell as the histamine-synthesizing endocrine cell in the rat stomach. Rat gastric oxyntic glands contain argyrophil "enterochromaffin-like" endocrine cells that synthesize and store histamine and also have APUD ability--they can take up exogenous L-5-hydroxytryptophan (5-HTP), can decarboxylate it to 5-hydroxytryptamine (5-HT, serotonin) by the enzyme DOPA-decarboxylase, and can store the amine. Previous cytochemical studies suggested that these cells correspond to both the ECL and A-like cells, the two predominant endocrine cells identified by electron microscopy (EM) in rat oxyntic glands. In a recent study, however, we demonstrated that the ECL but not the A-like cell exhibited APUD ability when rat gastric mucosa was incubated with H3-5-HTP and studied by EM radioautography. The purpose of the present study was to identify by EM radioautography the histamine-synthesizing endocrine cells in the rat stomach. Pieces of rat (male Sprague-Dawley) gastric mucosa were incubated in organ culture with L-H3-histidine (50 muCi, 1.8 x 10(-5) M) with and without inhibitors and were processed for LM and EM radioautography. H3-histidine labeled the ECL cells heavily but the A-like and other endocrine cells only lightly. The labeling of ECL cells was only modestly reduced by cycloheximide, an inhibitor of protein synthesis, whereas the labeling of A-like and other endocrine cells was almost abolished. In contrast, the labeling of ECL cells was markedly reduced by 4-bromo-3-hydroxybenzyloxyamine (NSD-1055), an inhibitor of histidine decarboxylase and DOPA decarboxylase, but was not appreciably affected by carbidopa, an inhibitor of only the DOPA decarboxylase. Incubations with H3-histamine (50 muCi, 0.9 x 10(-5) M) failed to label endocrine cells. Thus, this study demonstrates that the ECL but not the A-like cell is the histamine-synthesizing endocrine cell of the rat stomach.


Prifinium bromide in the treatment of the irritable colon syndrome. Eighteen patients with irritable colon syndrome were treated with a new anticholinergic drug (prifinium bromide) and with a placebo in a 6-wk, randomized, double-blind cross-over study. The drug was orally administered in a daily dose of 90 mg before meals. Three manifestations (pain, flatulence, constipation, and/or diarrhea), scored weekly, were used as assessment criteria. Mean over-all ratings showed a difference in favor of the drug, and were statistically significant. Side effects were rare and mild. We have come to the conclusion that this anticholinergic drug may be of benefit to patients with pain-predominant forms of irritable colon syndrome.


[Therapy with antacids].

[Therapy with antacids]. The usefulness of antacids in peptic ulcer disease is based on their ability to neutralize secreted gastric acid and to inhibit pepsin activity. The neutralizing capacity (expressed as mval of neutralized hydrochloric acid at a given pH) of different antacids depends upon their chemical composition. To achieve adequate neutralization of stimulated gastric acid secretion antacids have to be taken one and three hours after a meal in a dosage neutralizing 40-80 mval of acid. So far antacids have not been shown to be more effective in relieving ulcer symptoms than placebo. On the contrary an adequate antacid regimen will promote the healing of both gastric and duodenal ulcers in outpatients. Furthermore antacid prophylaxis will reduce the occurrence of acute gastrointestinal bleeding in critically ill patients.


The use of minor tranquilizers in a community mental health center. A study of 306 adult psychiatric outpatients visiting a CMHC during one month revealed that 55 per cent of those patients received a psychotropic medication and 45 per cent of the medication recipients used a minor tranquilizer as all or part of their treatment regimen. The most common diagnosis for patients receiving minor tranquilizers was depressive neurosis. The tranquilizers were prescribed in relatively high doses, and 57 of the 77 patients who recieved them had taken the drugs continuously for at least six months. The study also revealed that polypharmacy was widely practiced. The authors' suggestions for improving the quality of pharmacotherapy in a CMHC include instituting peer review of prescribing and recordkeeping practices and monitoring the effectiveness of medications through the application of quantitative mood scales at regular intervals.


Technologists look ahead to food safety. The institute of Food Technologists' meeting focused on food safety and looked at innovations taking place in equipment, processing, and energy conservation.


Continuous culture of normal adult mammalian hepatocytes exhibiting parenchymal functions. Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [3H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tryosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics.


Selection of a dexamethasone-resistant H-4-IIE-C3 rat hepatoma tissue-culture line. Exposure of H-4-IIE-C3 rat hepatoma cell cultures to the synthetic glucocorticoid, dexamethasone, results in an inhibition of cellular proliferation which is not the result of steroid-induced cytolysis. A significant decrease in both the rate of DNA synthesis and DNA content precedes a detectable effect on cell number. Continuous culture of H-IIE-C3 cells in medium containing 10(-5) M dexamethasone results in the selection of a steroid-resistant cell population that has the growth characteristics of unselected sensitive cultures and shows normal steroid induction of tryosine transaminase. Selection is a slow process requiring 24 to 36 months to obtain a phenotypically stable resistant cell line, and can be subdivided into three phases--a sensitive phase, adaptation and resistance. A comparison of the karyotypes of unselected and resistant cultures shows that the selection process enriches for a dexamethasone-resistant subpopulation.


Putrescine-oxidase activity in adult bovine serum and fetal bovine serum. Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.


[Animal experimental studies on partial kidney function after temporary tourniquet ischemia with and without blood substitute therapy]. 70 rats were subjected to tourniquet ischemia of a hind limb for a period of two and three hours. 12 rats served as controls. After release of the 3 hours tourniquets 20 rats were treated either with Ringer's solution or with hydroxyethyl starch. Kidney function and morphology, systolic blood pressure, hematocrit, serum electrolytes, creatinine and urea were studied as different times of recirculation. 1. Reduction in renal function was only observed after releasing the tourniquets. 2. The extent of reduction in renal function depended on the time of ischemia and time of recirculation. 3 hours of tourniquet with two hours of recirculation led to the largest extent of reduction in renal function and renal parenchymal lesions. 3. If the infusion of HES was applied at the beginning of recirculation, reduction in renal function was prevented, as well as parenchymal lesions. Ringer's infusion, however, did not improve kidney function to a normal range.


Interaction of pneumococcal antigens with complement in rats. Complement activation with pneumococcal antigens was studied both in vitro and after injection of the antigens into rats. Whole pneumococci of various serotypes activated C3-C9 in rat serum treated with ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, although serotypes differed greatly in the extent of activation. Some purified pneumococcal capsular polysaccharides also activated C3-C9 in rat serum, but only when the antigens were present in concentrations of 500 to 1,000 mug/ml. Much of the activation with capsular polysaccharides was eliminated by the use of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Activation of C3-C9 by capsular polysaccharides did not correlate with the level of reactivity observed with whole organisms of the same serotypes. After injection of 5 x 10(9) pneumococci (type 3 or type 4) intravenously into rats, there was a transient decline in serum C3-C9 activity, but there was no decline in C3-C9 levels after intravenous injection of 1,000 mug of type 3 or type 4 capsular polysaccharides. As determined by immunofluorescence, circulating capsular polysaccharide was deposited in several tissues, including the vascular endothelium and glomerular mesangium of the kidney. C3 was not detectable in these deposits, and there was no histological evidence of an inflammatory response. Capsular polysaccharides appear to be only weak activators of complement. Other pneumococcal antigens may be more important in the pathogenesis of hypocomplementemia in pneumococcal infection.


Factors affecting release of heat-labile enterotoxin by enterotoxigenic Escherichia coli. Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21 degrees C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37 degrees C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin.


Modification of antibody response to type III pneumopolysaccharide by route of injection of pertussis vaccine. Pertussis vaccine (PV) or diphtheria toxoid-PV-tetanus toxoid (DPT) altered the antibody response of BALB/c female mice to type III pneumococcal polysaccharide antigen (S3). The key factor affecting the magnitude of the response to S3 was the route of injection of PV or DPT, whereas the route of injection of S3 was not crucial. Subcutaneous injections of DPT augmented the antibody response to low, optimal, and tolerogenic (high) doses of S3 injected either subcutaneously or intraperitoneally. This enhancement was persistent and was observed both when S3 and DPT were mixed and injected subcutaneously and when S3 and DPT were injected concurrently at separate subcutaneous sites. When either PV or DPT was injected intraperitoneally, the antibody response to subcutaneously or intraperitoneally injected S3 was significantly decreased. These experiments demonstrate a dichotomy of effect dependent on the route of administration of PV. Most studies in mice utilizing PV employ the intraperitoneal injection route, and it is important to consider whether PV treatment by this route may have unique effects. Our data suggest that intraperitoneal injection of PV suppresses B cells, possibly by influencing regulatory cell function.


Clearance of bacteria from cerebrospinal fluid to blood in experimental meningitis. The occurrence and importance of secondary bacteremia in the pathogenesis of and response to therapy in meningitis is uncertain. Streptococcus pneumoniae type III was injected into the cerebrospinal fluid of the cisterna magna in anesthetized, curarized dogs, and sequential simultaneous samples were obtained from the superior sagittal sinus, cisterna magna, and peripheral blood. The results show that: (i) bacteria are rapidly transported from the cerebrospinal fluid to blood but only after active multiplication within the cerebrospinal fluid, and (ii) entrance into the blood from the cerebrospinal fluid occurs before the height of the febrile response or cerebrospinal fluid pleocytosis.


Superoxide production in pulmonary alveolar macrophages and killing of BCG by the superoxide-generating system with or without catalase. The superoxide production of BCG-infected and noninfected alveolar macrophages was measured by superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The cells were incubated with or without cell-free bronchial lavage fluid (pulmonary washings). When control alveolar macrophages were infected by BCG, superoxide production was decreased markedly, probably due to bacterial cytotoxic factors. In contrast, the production of superoxide in alveolar macrophages exposed to pulmonary washings was increased and not appreciably influenced by BCG infection. Superoxide production by alveolar macrophages was dependent on time and on the protein concentration in the pulmonary washings. In controls, it was inversely proportional to the infecting dose of BCG. We observed previously that alveolar macrophages activated by pulmonary washings inhibited intracellular growth of BCG. We now present evidence that enhanced production of superoxide contributes to such inhibition, especially in the presence of catalase at acid pH. These findings are pertinent to the defense of inflamed lungs, where serum and serum immunoglobulin G transuded from blood into alveolar spaces probably induce such activation on alveolar macrophages.


Purification and characterization of a Serratia marcescens metalloprotease. An extracellular, nonelastolytic, neutral metalloprotease of Serratia marcescens was purified by sequential ammonium sulfate precipitation, hydroxyapatite adsorption chromatography, flat-bed isoelectric focusing, and Sephadex G-100 gel filtration. The protease preparation had a 280/260 nm absorbance ratio of 1.8, was free of detectable amounts of endotoxin, carbohydrate, phosphorus, and other known extracellular enzymes of S. marcescens, and was homogeneous by Ouchterlony double immunodiffusion and Grabar-Williams immunoelectrophoresis. Crossed immunoelectrophoresis, thin-layer electrofocusing in polyacrylamide gel, and polyacrylamide disc gel electrophoresis showed three to four closely migrating, Coomassie blue-staining components in the protease preparation. However, zymogram analyses of the patterns showed that protease activity was associated with each component and that the protease was, therefore, microheterogeneous. The isoelectric point and sedimentation coefficient of the protease were approximately 5.3 to 5.4 and 4.2S, respectively, and the molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration was approximately 52,500 and 44,000, respectively. The pH optimum range, with azocasein as the substrate, was 5.5 to 7.5. The enzyme contained a high percentage of acidic amino acids, no cysteine, and 1 g-atom of Zn(2+) and 7 g-atoms of Ca(2+) per mol. Various heavy metal ions and chelating agents and heating at 60 degrees C for 15 min inactivated the enzyme. Intracorneal, intratracheal, and intradermal administration of the protease into rabbits elicited rapid and extensive tissue damage. The minimum lethal intravenous dose for mice was approximately 17 mg/kg of body weight.


Purification and properties of rabbit alveolar macrophage lysozyme. Lysozyme was isolated from Bacillus Calmette-Guerin-elicited rabbit alveolar macrophages by acid extraction and purified to homogeneity by a single-column procedure. Yields of the purified enzyme averaged between 20 and 30 mg per rabbit, values far in excess of those obtained with previously published methods. Rabbit lysozyme has a molecular weight of 14,300 and exhibits optimal lytic activity against Micrococcus lysodeikticus at an ionic strength of 0.04, pH 6.5. Our results indicate that lysozyme and other granule components can be fractionated from elicited alveolar macrophages by using simple techniques, suggesting methods for the bulk purification of lysosomal constituents.


Antibacterial activity of bladder surface mucin duplicated by exogenous glycosaminoglycan (heparin). We have previously shown that the transitional cells lining the urinary bladder are capable of producing glycosaminoglycan (GAG). By use of a quantitative in vivo method of measuring bacterial adherence, we demonstrated that bacterial adherence to the mucosal cells is diminished in the presence of this GAG, rises when it is removed (by acid), and returns to normal when the GAG is resynthesized (in less than 24 h). We also found that this much layer could be removed (with a corresponding rise in bacterial adherence) and that addition of exogenous GAG (heparin) to the bladder prevented the expected rise in bacterial adherence. This study analyzed in depth the manner by which heparin prevents the rise in adherence seen when the mucin is removed. Pretreatment of bacteria with heparin had no effect on adherence, whereas pretreatment of the bladder with heparin inhibited adherence. To corroborate our impression that the heparin was coating the transitional cells, [3H]heparin was added to bladders after removal of mucin. Autoradiography revealed the heparin to be adherent to the surface of the transitional cells.


Risk factors for cancer of the testis in young men. An individual matched case-control study of testis cancer in 131 men under age 40 was conducted to investigate antecedent risk factors including events during prenatal life. Ten patients were born with an undescended testis compared to only two controls (p less equal to 0.02), a previously reported risk factor. Two new risk factors were uncovered: six patients-mothers received hormones during the index pregnancy compared to only one control-mother, and eight patient-mothers and two control-mothers reported excessive nausea as a complication of the index pregnancy. A hypothesis linking these three factors is presented: viz, that a major risk factor for testis cancer is a relative excess of certain hormones (in particular estrogen) at the time of differentiation of the testes.


Characterization of sperm agglutinins in sera from infertile women. Sperm agglutinins detectable by a tray agglutination technique in sera from women from infertile couples were characterized. Preparative zone electrophoresis of seven head-to-head-agglutinating sera revealed two patterns. In two sera sperm agglutinins were obtained in the gamma-globulin fraction, whereas in five sera the agglutinating activity was detected in the beta-globulin fraction only. By immunoaffinity chromatography, IgM and IgG sperm antibodies were demonstrated in the former two sera and also in two tail-to-tail-agglutinating sera. The beta"sperm agglutinin was not found to share physicochemical or immunological characteristics with beta-lipoprotein in the present investigation by ultracentrifugation, by precipitation of beta-lipoprotein with heparin and MnCl2, by absorption of beta-lipoprotein with colloidal silic acid, or by affinity chromatography on an anti-beta-lipoprotein column. An apparent neutralizing effect of anti-beta-lipoprotein by simple addition of antiserum to sera containing the beta-sperm agglutinin was shown to be due to a reaction with sperm components released from the spermatozoa by which the antiserum had been absorbed before use. These sperm components were found to neutralize the activity of the beta-sperm agglutinin only, whereas no effect was observed in sera containing sperm antibodies. By Sephadex G-200 fractionation the beta-sperm agglutinin was shown to have a high molecular weight (MW greater than or equal to 600,000). Ultrafiltration and dialysis experiments gave no evidence of involvement of low-molecular compounds in head-to-head agglutination.


Change in the classic gynecologic surgery: review of 3,300 pelviscopies in 1971-1976. An endoscopic microsurgical technique has been developed on the basis of the development of a new technique for hemostasis (endocoagulation), which needs neither ligation nor high-frequency current, and a specific pelviscopic instrument setup for surgical therapeutic pelviscopy has been created. This new endoscopic technique, i.e., the microlaparotomy via pelviscopy, opens new methods for pelvic surgery, in particular for patients with infertility. Our data show that in 79% of tubal occlusions, patency can be achieved by pelviscopic surgery. In addition, lysis of intestinal adhesions, tubal sterilizations, endometriosis-coagulation, ovarian cyst resection, subserous myomectomy, and total oophorectomy can be performed with this newly devised procedure. Our data show that in our series of 3,300 pelviscopies, the complication rate was 1.1%, and only 0.1% necessitated laparotomy. In 55%, pelviscopy performed after previous laparotomies added very little risk. In 61%, destructive heat was utilized for hemostasis. No intra-abdominal burns occurred in the entire series of 3,300 patients. With this new method, when used for protein-coagulation to secure hemostasis, not one burn occurred.


Comparison of the lytic effects of four prostaglandin analogues in the chacma baboon (Papio ursinus ursinus). Six healthy, cycling female chacma baboons (Papio ursinus ursinus) were used to determine the luteolytic effects of four prostaglandin analogues. The compounds were administered according to a study design which made provision for adequate controls. Five to seven days following ovulation, venous blood was collected and the baboon given a single intramuscular injection of a compound at a recommended dose. Blood was then collected serially every 3 h for three samples and again at 24, 48, and 72 h to determine the continued effect of the prostaglandin analogues on corpus luteum production of progesterone. It was found that PGF2alpha-1,15-lactone, 11alpha(15S)-17-phenyl-18,19,20-trinor-ent-PGE2 methyl ester and 17-phenyl-18,19,20-trinor-PGF2alpha exhibited definite luteolytic potential in this species. Equivocal results were obtained with (15S)-15-methyl-PGF2Alpha THAM and its toxic qualities resulted in the demise of three animals.


Effect of caffeine and kallikrein on cryo-preserved human spermatozoa. Preservation of human semen in liquid nitrogen causes a significant impairment of sperm motility. Ejaculated human spermatozoa show an increased motility in the presence of caffeine, a phosphodiesterase inhibitor, and pancreatic kallikrein (EC 3.4.21.8), a kinin-producing proteinase. Hence, the effect of both substances on post-thaw motility, fructose consumption, and cervical mucus penetration of cryo-preserved human spermatozoa was investigated. The results indicate that both substances stimulate the motility of freshly ejaculated spermatozoa and also improve the motility pattern of cryo-preserved human spermatozoa, thus offering a possible means of improving the quality of freeze-preserved human semen.


Sensitivity of human spermatozoa to various isolation procedures: differences in relation to sperm counts. The aim of the present work was to examine the sensitivity of human spermatozoa from oligospermic and normospermic semen samples to manipulations in the course of isolation by generally used procedures. To this end we performed protein and DNA estimations in spermatozoa isolated by procedures differing in rigorousness (washings with Ringer's buffer and a medium containing ficoll) as well as microscopic examinations of washed sediments. The results showed that the washed sediments contained, in addition to spermatozoa, materials containing protein and DNA, possibly deriving from the breakage of sperm cells. These materials increased in amount with the decrease of original sperm counts. We suggest that abnormal spermatozoa, whose percentage usually increases with the severity of oligospermia, are more sensitive to damage than the normal ones.


The biological activities of norgestrel and its enantiomers. The dextrorotatory enantiomer of norgestrel has now been tested by oral and parenteral routes in a broad range of biological assays and its inactivity has been confirmed. The active enantiomer has been further compared with the racemate in a similarly broad range of tests, largely by the oral route. Levonorgestrel was twice as potent as the racemate in experiments in which norgestrel was effective. These data reinforce earlier findings that levonorgestrel is responsible for all the biological activity found in norgestrel.


Isolation of human spermatozoa membrane antigens binding sperm-immobilizing and sperm-agglutinating antibodies. Peptides of human spermatozoa were dissolved with Hyamine 2389 and Triton X-100 and separated by chromatography on Biogel P4 columns into seven fractions. The antigenic activities of the separated sperm-membrane fractions were tested according to their capacity to inhibit sperm agglutination and sperm immobilization (immune inhibition test) in human sera of sterile patients. Sperm-agglutinating and sperm-immobilizing activity was tested by the microtray agglutination and microtray immobilization test. A titer reduction was achieved only in sperm-immobilizing sera. Four sperm antigenic fractions revealed in the majority of the repeatedly tested sperm-immobilizing sera an inhibition of the antigen-antibody reaction. No reaction was observed after exhaustive absorption of the tested seven antigen fractions with sperm-agglutinating sera. Therefore the conclusion can be drawn that sperm-agglutinating and sperm-immobilizing antibodies react with different sperm antigens. Normal human sera without sperm antibodies served as control. As no sperm agglutination or sperm immobilization was obtained after absorption of these control sera our antigen fractions do not produce sperm agglutination or sperm immobilization.


A new technique for intrauterine insemination. The 5-cm long endpiece of a soft Nelaton's catheter, ch 6-8, splinted with a rigid polyethylene cannula, proved to be a suitable instrument for intrauterine homologous inseminations. Sixty couples were treated; indications for intrauterine insemination were subfertile semen, poor postcoital test, antisperm antibodies in male or female, and unexplained infertility of more than 5 years. Seventeen pregnancies occurred, that is 28%. The best results were obtained in the group with unexplained infertility and in the group with poor postcoital tests, combined with a good result of the in vitro sperm penetration tests. Complications of the inseminations were rare. The method did not elicit sperm agglutinins in the female; however, if sperm agglutinins were already present in the female, the titre level increased after the intrauterine inseminations.


Prolactin levels and bromocriptine treatment of short luteal phase. Moderate hyperprolactinemia was found in 14 of 30 infertile patients with short luteal phase indicating a possible hypothalamic disorder in these patients. While the cycle length was normal, 28 days, late ovulation around day 18 of the cycle was characteristic of these patients. During bromocriptine treatment, 2.5 mg twice daily, ovulation took place earlier and luteal phase became longer irrespective of the basal serum prolactin level. The mean (+/- SEM) duration of luteal phase was 9.9 +/- 0.2 days in control cycles, and 11.7 +/- 0.5 and 12.2 +/- 0.3 days in two successive bromocriptine cycles (P less than 0.001). In patients taking bromocriptine, luteal phase became longer than 11 days in 37 of 60 treatment cycles, but no significant difference was recorded in the circulating progesterone and LH levels during mid- and late luteal phase. Three patients became pregnant and they all had normal baseline serum prolactin concentrations. Our results show that bromocriptine may be effective even when no apparent indication for prolactin suppression can be demonstrated.


Chemical inducers of ovulation: comparative results. Chemical inducers of ovulation are frequently used to reestablish a normal hypothalamic-ovarian function in the sterile woman. At present, several types are available and it is useful to evaluate comparatively their efficacy. In this paper we present our results in 396 cases treated with some of these drugs. Clomiphene citrate was administered to 307 patient. Ovulation was obtained in 85.99% and pregnancy in 35.50%. There were 16 abortions (14.68%) among the 109 pregnancies obtained. Cisclomiphene was used in 11 cases. The ovulation rate was 81.81%, with 54.5% of pregnancies. Thirty-eight patients were treated with Cyclophenil. Ovulation was obtained in 71.05% of the cases and pregnancy in 23.68%. In forty cases Tamoxifen was administered: the ovulation rate was 95% and the pregnancy rate was 35%, but the abortion rate was 35%. Tolerance was good with all medications. They had similar adverse side effects (mild and rare). None produced overstimulation. With Cyclophenil and specially with Tamoxifen the cycles were longer. The incidence of abortion in the 131 pregnancies obtained was 14.68% with Clomiphene; none with Cisclomiphene; 11.11% with Cyclophenil, and 35.10% with Tamoxifen. We conclude that Clomiphene is the drug which gives  the best results at present.


Is conception in infertile couples treatment-related? A survey of 309 pregnancies. 309 couples with infertility of 1 to 11 years duration in whom pregnancies occurred during examination or treatment were evaluated. The couples were classified into diagnostic groups according to the main cause of their infertility; anovulatory, mechanical, male, cervical, unexplained, combined. Pregnancies were divided into treatment-related and nontreatment-related. Analysis of results revealed that whereas in infertile patients with a defined cause of infertility, the treatment-related pregnancy rate was 73.1%, in the unexplained infertility group the incidence of treatment-related pregnancies was only 6.6%. We concluded that a meticulous fertility survey should be carried out in each infertile couple in order to detect a specific cause impeding fertility, but if such a cause cannot be found, reassuring of patients and decreasing their anxiety is more effective in inducing pregnancy than nonspecific therapy.


Infrequent structural chromosome aberrations in women with primary amenorrhea. Since the problem of primary amenorrhoea (PA) is very real the authors reviewed a series of 58 women from their own laboratory, citing all known causes of PA and emphasizing that the most interesting cases are those with normal karyotype. Reinvestigation of patients with normal karyotype using the banding methods (G and R) revealed new types of structural aberrations of X chromosome. The importance of structural disturbances and rearrangements of particular segments of X chromosome in the etiology of PA has been discussed.


Effect of synthetic LH-RH and hCG administration on plasma testosterone, androstenedione, and estradiol 17beta levels in normal men and in patients with idiopathic oligospermia. Plasma testosterone, androstenedione, and estradiol 17beta levels were measured in 20 normal men and in 20 patients with idiopathic oligospermia before and after iv hCG (5,000 IU) and synthetic LH-RH (50 microgram) administration. No statistically significant differences in the concentrations of the steroids examined were detected between patients and controls either under control conditions or under exogenous and endogenous gonadotropin stimulation.


Effect of acebutolol on dynamic ischemic ECG changes: a study using ambulatory twenty-four-hour ECG monitoring. The clinical effect of the beta-adrenergic blocking drug acebutolol hydrochloride (SECTRAL) was studied in 18 patients with angina pectoris. Ambulatory 24-h ECG monitoring proved to be a useful method for assessing the efficacy of this drug in individual patients in their own daily stresses and environment. Patients were studied in the control state, after two weeks' treatment with placebo, and after two weeks' constant oral dose of the drug. It was found that acebutolol produced a significant decrease in ischemic ST segment depression in patients in whom good beta-blockade was achieved. The drug was less effective in patients in whom the heart rate response to exercise was not suppressed and in those with critical coronary artery obstructions.


Oxygen binding of fetal and adult bovine hemoglobin in the presence of organic phosphates and uric acid riboside. The P50 values of "stripped" fetal and adult bovine hemoglobin were 18.4 and 28.9 respectively. Neither the oxygen-hemoglobin dissociation curve nor the Hill coefficient, n, of fetal or adult bovine hemoglobin was affected by uric acid riboside (UAR), 2,3-diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), or inositol pentaphosphate (IPP). Combinations of UAR and ATP with adult bovine hemoglobin or 2,3-DPG and ATP with fetal hemoglobin also had no effect. It was concluded that neither adult nor fetal bovine red cells contained an identifiable compound which affects the binding of oxygen to hemoglobin.


A cytochemical study of the localization of acid phosphatase in Saccharomyces cerevisiae at different growth phases. The localization of acid phosphatase in the yeast Saccharomyces cerevisiae at different growth phases had been studied. It was shown to be crucial for authentic location of acid phosphatase that the cytochemical reaction be performed on whole cells. Dimethylsulphoxide was used to alleviate the effects of fixation of the yeast cells with glutaraldehyde; the sulphoxide did not affect the distribution of acid phosphatase in the cells. It has been established that in exponentially-growing cells acid phosphatase is localized mostly in small vacuolar compartments. In mature cells, the bulk of acid phosphatase is found in the central vacuole, although a significant amount of the enzyme is detectable in the plasma membrane and the adjacent vesicles.


Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits. Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.


Adaptations in skeletal muscle following strength training. Five men were studied before and after 7 wk of isokinetic strength training to determine its effects on muscle enzyme activities and fiber composition. One of the subject's legs was trained using 10 repeated 6-s maximal work bouts, while the other leg performed repeated 30-s maximal knee extension exercise. The total work accomplished by each leg was constant. Training 4 times/wk achieved similar gains in peak torque for both legs at the training velocity (3.14 rad/s) and at slower speeds. Fatigability of the knee extensor muscles, as measured by a 60-s exercise test, was similar in both legs after training. Biopsy specimens showed significant changes in the % of the muscle area composed of type I and IIa fibers as a result of both strength training programs. In terms of muscle enzymes, only the 30 s exercise program resulted in elevated glycolytic, ATP-CP and mitochondrial activities. Despite these changes, none of the parameters measured were found to be related to the gains in either muscle strength or fatigability during maximal isokinetic contractions.


A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. I. Fermentation, isolation and characterization. Two new aminoglycoside antibiotics, sporaricins A (C17H35N5O5) and B (C15H32N4O4) produced by a rare actinomycetales, Saccharopolyspora hirsuta subsp. kobensis have been isolated by column chromatography on a cation-exchange resin. Sporaricin A is highly active against Gram-positive and Gram-negative bacteria including aminoglycoside-resistant strains.


Purification and partial characterization of an antibiotic produced by Myxococcus coralloides. A strain of Myxococcus coralloides producing an antibiotic capable of inhibiting growth of Gram-positive bacteria was isolated. Antibiotic production occurred during vegetative growth but not during myxospore formation. The antibiotic was extracted from the growth medium with chloroform and purified by adsorption on silicic acid and by preparative silica gel thin-layer chromatography. The purified antibiotic showed a resistance to heat, acid, alkali and proteolytic enzymes. Chromatographic and electrophoretic behavior as well as infrared, ultraviolet and mass spectra are presented.


Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides. Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.


Phosphate-limited continuous culture of Rhodotorula rubra: kinetics of transport, leakage, and growth. The phosphate-limited growth kinetics of Rhodotorula rubra, a small yeast of marine origin, were examined by analysis of 32P distributions in continuous cultures. Isotope relaxation procedures were used to identify unidirectional flows of Pi and organic phosphate among compartments modeled during growth. The concentrations of phosphates in these compartments at various growth rates were used, together with attendant flows, to produce a mathematical model of growth. Both Pi and phosphate-containing metabolic intermediates leaked from cells during growth. Total leakage ranged from 4 to 10% of influx and was comprised mostly of Pi. Transport capacity was at least 10 times that required for growth at saturating Pi concentrations, so that influx was linear with concentration during growth. This led to the realization that the curvature of Monod plots (Kmu = 12 nM mumax = 0.18/h, and the threshold At = 2.5 nM) is due to change in yield with growth rate. Growth rate related to Pi by the affinity, aA (= 0.43 liter/mg of cells.h) of cells for Pi and the growth rate-dependent yield. It was also specified by a series of kinetic constants that specified flow among the various compartments and equilibrium compartment concentrations as they were set by extracellular Pi. The importance of leakage by healthy cells to the organic chemistry of aquatic systems is noted.


Enhanced transformability with heterospecific deoxyribonucleic acid upon removal of nascent ribonucleic acid from the Streptococcus sanguis genome. Treatment of Streptococcus sanguis recipient cells with rifampin (RIF) at the time of deoxyribonucleic acid (DNA) addition was an effective means of reducing discrimination, that is, of causing an increase in the number of transformants induced by irreversibly bound heterospecific DNA without significantly changing the number induced by bound homospecific DNA. RIF was unable to reduce discrimination when the recipient cells were RIF resistant due to an altered ribonucleic acid (RNA) polymerase. When recipient cells were treated at the time of DNA addition with concentrations of streptolydigin (STG) as inhibitory of RNA synthesis as RIF, discrimination was not reduced. The kinetics of RNA synthesis inhibition with these inhibitors indicated that, as reported for other bacterial species, RIF inhibited the initiation of transcription by RNA polymerase, whereas STG inhibited the progression of RNA polymerase at any point. Pulse-labeling of RNA immediately before STG addition showed that, if cells were incubated under STG inhibition for 10 to 15 min, their nascent RNA was degraded. Genome-bound RNA polymerase was not released under these conditions. When recipient cells were incubated with STG until nascent RNA was degraded and then exposed to transforming DNA, STG was as effective as RIF in reducing discrimination. The presence of nascent RNA was thereby implicated in the transforming inefficiency of incompletely homologous DNA.


Structure of common pili from Escherichia coli. Several important properties of the common pili from Escherichia coli are discussed. These pili were resistant to the gentle Folin-Ciocalteau reagent methods for protein detection and were not readily solubilized by sodium dodecyl sulfate. They were found to contain a reducing sugar but not peptidoglycan. The pilin had multiple conformations in sodium dodecyl sulfate solution, and the appearance of multiple bands on sodium dodecyl sulfate gels did not necessarily indicate heterogeneity of the preparation. The ilus subunit was found to be a different protein than outer membrane III, which has the same apparent molecular weight. In addition, we conformed the results of Brinton (Trans. N.Y. Acad. Sci 27:1003-1054, 1965): that there is a dramatic change in the properties of pili after they are heated at pH values below 2.


Reduction of methionine sulfoxide to methionine by Escherichia coli. L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.


Growth of Nocardia rhodochrous on acetylene gas. Soil sediment enrichment cultures yielded a coryneform bacterium capable of growing in a mineral salts solution with acetylene gas as its only source of carbon and energy. Based on morphological and physiological traits as well as on cell wall analysis, the bacterium was characterized as a strain of Nocardia rhodochrous. Maximal growth rates (generation time 2.7 to 3.0 h) on acetylene were obtained at 5 to 20% acetylene, 25 to 40% oxygen, pH 7.0 and 26 to 28 degrees C. Yields (grams of dry cells produced per gram of acetylene consumed) ranged between 90 and 110%. N. rhodochrous exhibits a growth factor requirement for the pyrimidine moiety of thiamine. Acetylene utilization is not an obligate trait, and a wide range of alternate carbon sources is utilized. Ethylene is neither produced nor consumed. The only previous report on acetylene utilization appeared in 1932. The Mycobacterium lacticola strain described in that report strongly resembles N. rhodochrous.


Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R. Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.


Effect of cyclic guanosine 3',5'-monophosphate on nitrogen fixation in Rhizobium japonicum. The addition of exogenous cyclic guanosine 3',5'-monophosphate (cGMP) at a concentration of 0.1 mM to a free-living culture of Rhizobium japonicum 3I1b110 was found to completely inhibit the expression of nitrogenase activity and markedly inhibit the expression of hydrogenase and nitrate reductase activities. The effect was specific for cGMP. Experiments on the in vivo incorporation of radioactive methionine and subsequent analysis of the labeled proteins on polyacrylamide gels showed that the biosynthesis of nitrogenase polypeptides was inhibited. It appears that the time of addition of cGMP is important since the effect was only seen during the early stages of nif gene expression. The intracellular level of cGMP was found to respond to physiological changes in the cell, and there was a fall in cGMP concentrations when nitrogenase was induced. Microaerophilic-aerobic shift experiments showed that intracellular levels increased from 0.25 pmol/mg of cell protein under microaerophilic conditions to 2.6 pmol/mg of cell protein under aerobic conditions, suggesting that the cellular pool size of cGMP may be under redox control.


Organization and transfer of heterologous chloramphenicol and tetracycline resistance genes in pneumococcus. The cat and tet genes of chloramphenicol- and tetracycline-resistant clinical isolates of Streptococcus pneumoniae from Paris and Japan were shown to be contained in adjacent heterologous insertions into the chromosome. The two insertions transformed laboratory strains at frequencies that were low, unequal, and, for tet, very sensitive to the length of the donor deoxyribonucleic acid strand. In contrast, the transforming activity of cat was relatively stable. There was an unusual asymmetric cotransfer, in that a majority of the tet transformants also acquired cat, whereas only a few of the cat transformants also acquired tet. The evidence for chromosomal insertion came from genetic data showing linkage of cat to a chromosomal gene and from cosedimentation of cat with chromosomal markers in both velocity and dye-buoyancy experiments. Genes on a known plasmid introduced into pneumococcus from Streptococcus faecalis showed very different physical behavior. Most of the transformation properties of these genes can be readily accounted for by analogy to transformation of deletions of normal genes. Whether transposition contributes any of the transfers remains to be determined. The presence of one of the genes in the recipient promoted the integration of the other, demonstrating enhanced accumulation of heterologous genes by a process that did not involve plasmids in the species of concern.


Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs. Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.


Role of lithium ions in proline transport in Escherichia coli. Mechanisms of Li+ stimulation of proline transport were studied in cells of Escherichia coli 7 and NR70, a mutant of strain 7 lacking adenosine triphosphatase (EC 3.6.1.3). An electrochemical potential difference of Li+ induced in an inward direction of energy-depleted cells caused a transient uptake of proline depending on the driving force provided. When proline was added to unbuffered cell suspensions under anaerobic conditions, the medium was found to be acidified only in the presence of Li+ but not in the presence of Na+ or K+. This acidification was abolished by the addition of a permeant anion, SCN-, to the medium containing Li+, but this was not demonstrated with cells of a mutant strain deficient in a carrier protein specific for proline. These results support  the assumption that proline is taken up by a mechanism of Li+-proline cotransport in E. coli.


Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics. Two mutants (NT02 and NT17), each producing a thermosensitive neutral protease, were isolated from Bacillus subtilis NP58, a transformant which acquired the property of hyperproduction of neutral protease from Bacillus natto IAM 1212. The neutral proteases produced by these two mutants were partially purified and enzymologically characterized. The two mutant neutral proteases displayed increased thermosensitivity as well as altered pH optima compared with those of the NP58 enzyme. In addition, the hydrolytic activity of the thermosensitive neutral proteases on synthetic peptide substrates was found to be extremely different. These results strongly suggest that the site of mutation in each of the temperature-sensitive strains is located within the structural gene for neutral protease (nprE). Previous studies indicated the existence of a specific regulator gene (nprR) in addition to the structural gene for neutral protease. Phage PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation studies with the parental and mutant strains suggest that the chromosomal order of these genes is recA-pyrA-nprR-nprE-fruB-metC. Moreover, the results of these genetic analyses imply that the mutations to thermosensitivity are located proximate to each other within the nprE gene.


Regulation of Saccharomyces cerevisiae nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase by proteolysis during carbon starvation. Inactivation of the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase from Saccharomyces cerevisiae during carbon starvation occurs with a simultaneous loss of enzyme protein and enzyme activity.


Nitrogen metabolite repression of nitrate reductase in Neurospora crassa: effect of the gln-1a locus. Nicotinamide adenine dinucleotide phosphate (reduced form)-nitrate reductase was freed from ammonium repression in a Neurospora crassa mutant having drastically lowered glutamine synthetase activity, gln-1a. The general phenomenon of nitrogen metabolite repression required glutamine or some aspect of glutamine metabolism.


Effect of NADP+ and its analogs on the Rose Bengal-sensitized photoinactivation of D-erythrulose reductase from beef liver. Upon addition of NADP+, the rose bengal-sensitized photoinactivation of D-erythrulose reductase from beef liver is prevented to a remarkable extent. Adenosine 2',5'-diphosphate (2',5'-ADP) also has a protective effect, but to a lesser extent. On the other hand, 2'-AMP markedly enhances the photoinactivation. Other nucleotides which have no 2'-phosphoryl group, such as NAD+, 3'-AMP, 5'-AMP, ADP, and NMN, are ineffective. Further, only 2'-AMP derivatives (NADP+, 2',5'-ADP, and 2'-AMP) among these nucleotides were found to be potent competitive inhibitors of the enzyme with small Ki's (6--13 muM). Photooxidation of some methionine residues in the enzyme is prevented by the addition of NADP+ and accelerated in the presence of 2'-AMP. Photooxidation products(s) of 2'-AMP derivatives have no effect upon the enzymatic activity. Although NADP+ and 2'-AMP induce detectable conformational changes of the enzyme, the changes are not characteristic to the compounds. Based on these observations, we present a possible action mechanism of 2'-AMP derivatives on the photoinactivation of D-erythrulose reductase.


A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein. To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.


Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens. NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.