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Effect of catecholamines on oedema induced by inflammatory agents in the rat.

The effect of intracutaneous adrenaline and noradrenaline (5 X 10(-12) and 5 X 10(-11) mol) was examined on the oedema (Evans blue dye leakage) response of rats to several inflammatory agents. The catecholamines reduced the oedema response to all agents tested except prostaglandin E1 (PGE1) which was significantly potentiated by noradrenaline (5 X 10(-11) mol), and a combination of bradykinin 5 X 10(-11) mol with PGE1 5 X 10(-10) mol which was not significantly affected by any dose of catecholamine. Adrenaline was more effective than noradrenaline in reducing oedema produced by 5-hydroxytryptamine (5HT) and histamine and by agents which release these amines (compound 48/80, dextran and antigen challenge with egg albumin in sensitized rats), but noradrenaline was more potent against bradykinin-induced oedema. The inhibitory effect of catecholamines against oedema produced by histamine and 5HT was abolished by a combination of phentolamine and propranolol. It was concluded that the oedema-inhibiting effect of catecholamines is due to alpha- and beta-adrenoreceptor mediated actions.

[Clinical and immunological studies on acquired heat contact urticaria (author's transl)].

A case of localized urticaria in an otherwise healthy young woman, produced only by direct contact of the skin with heat, is described. The minimal temperature of urtication was 44 degrees C (immersion of the forearm in hot water for 5 min). Redness and painful oedema immediately developed without reflex flare. Total serum IgG, IgA, IgM, IgE, complement factors C3 and C4, and alpha1-antitrypsin were in the normal range, whereas the C1-inhibitor level was slightly decreased. There was no evidence of circulating immune complexes in the serum. A skin test and a RAST with house dust were positive, but there were no signs of respiratory atopy. An attempt for passive transfer of heat urticaria into the abdominal skin of a rhesus monkey failed, but was successful for house dust. A treatment trial with ketotifen, a new, perorally acting anti-allergic drug, was poorly effective, but dexchlorpheniramine maleate, a classical antihistaminic, in a dose of 12 mg daily completely suppressed the swelling evoked by heat, but not the erythema, suggesting that other tissue or plasma factors than histamine may be involved in the mechanism of heat urticaria in this patient.

Sterilization of women: benefits vs risks.

Voluntary sterilization is the birth control method most widely practiced throughout the world. The last ten years have witnessed great improvements in techniques and perfection of innovations, explaining the important role that it now plays in the regulation of fertility. Different methods are examined and it is concluded that hysterectomy is the best, if medically indicated; conventional laparotomy is not justified unless required by concomitant intraabdominal pathology; minilaparotomy is mostly suitable postpartum; colpotomy is better left to specialists; laparoscopy is ideal for nonpregnant patients; culdoscopy is a relic of the past; and hysteroscopy, although still experimental, may be the way of the future. The advantages of voluntary sterilization lie in its remarkable and immediate efficiency, freedom from ongoing motivation, the convenience of a one-time operation, the absence of side effects and the reduction of total costs. Its disadvantages are the complexity of any surgical intervention for a woman, its indisputable finality, its uncertain legality and the risks inherent in any operation. Hysterectomy and tubal ligation are practically never fatal, so this argument does not influence the choice of either method. However, incidence of morbidity is higher following hysterectomies, which must therefore be justified. The balance is clearly in favor of voluntary sterilization for the woman who is convinced that the size of her family is complete.

Biochemical basis for the selection of oral contraceptives.

Many specific plasma proteins show dose-related changes when oral estrogens are administered. Large increases in concentration are seen in many important binding proteins, such as the sex hormone-binding globulin, transcortin, the retinol-binding protein, ceruloplasmin, and transferrin. A smaller group of plasma proteins are reduced in amount. These changes are related to altered rates of hepatic synthesis and secretion. As the overall effect of estrogen is one of increased protein synthesis, there is a reduction in the amount of plasma-free amino acids and in the pattern of distribution. Oral contraceptive (OC) users frequently show significant alterations in biochemical tests of vitamin status, at least some of which are related to alterations in plasma proteins. Other biochemical changes associated with OC use include a fasting hyperlipidemia, due mainly to increases in triglycerides, although there is often also a small increase in cholesterol. These changes are due primarily to increases in several lipoprotein fractions and are related mainly to the estrogen component. A deterioration in glucose tolerance occurs in many OC users and is probably induced by both estrogens and progestogens. There is evidence that certain clinical side effects of OCs, such as depression, are associated with specific biochemical changes.

Sex differences in fetal sheep adrenal steroidogenesis.

The formation of several steroids was determined in vitro in adrenals removed from 18 female and six male fetuses of 113-115 days' gestation and in two female and two male fetuses at near term (137-143 days). Samples were incubated with 14C-acetate and the formation of labeled steroids was determined by two-dimensional paper chromatography. Protein and corticosterone concentrations were determined by chromophore absorption and acid hydrolysis fluorescence methods, respectively. Tissue corticosterone concentrations were significantly higher in female (0.145 +/- 0.010 microgram/mg protein) than in male (0.083 +/- 0.010 microgram/mg protein) adrenal tissue at both stages, whereas corticosterone formation was similar in both sexes. Cholesterol formation was significantly higher in female (0.103 +/- 0.079 muM/mg protein) than in male (0.044 +/- 0.011 muM/mg protein) adrenals at both stages. Both testosterone and estradiol were synthesized at higher rates in female than in male adrenals (52% and 33%, respectively), whereas pregnanediol formation was 21% higher in the male. These results indicate that significant sex differences exist in the formation of various adrenocortical hormones by fetal tissues. The relevance of these findings to better survival of female premature newborns from respiratory distress syndrome in contrast with male, is discussed.

Euhypnos Forte (temazepam) for resistant insomnia: a clinical trial.

Seventy insomniac patients, previously unresponsive to conventional hypnotic dosage, were treated for seven nights with temazepam in 20 mg soft gelatin capsules (Euhypnos Forte). The patients adjusted the dose to suit themselves up to a maximum of 60 mg. Nineteen patients found that one 20 mg capsule suited them best in spite of previous lack of response to two 10 mg capsules, and were thus excluded from the final analysis. Out of the remaining fifty-one patients, thirty-five were best suited by 40 mg and sixteen by 60 mg of temazepam. Sleep was rated Very Good or Good by forty patients (78.4%) and significant hangover occurred in only four (7.8%), all of whom were on 40 mg. Adverse reactions were insignificant. Some observations by one author (CALM) on the significance of the results in the management of insomnia in general practice are included.

Placental clearance of lactate and bicarbonate in sheep.

To determine the placental clearance of lactate and bicarbonate in sheep, the fetal side of an isolated cotyledon and the umbilical circulation of the total placenta were artificially perfused. The release and uptake of lactate and bicarbonate by the perfusion fluid and the fetomaternal concentration differences of these substances were measured. From these data, the clearance of lactate and bicarbonate was determined to be 0.9 (SE = 0.2) ml/h/g of placental tissue. The production of lactate by the placenta was calculated to be about 30 mumol/min, the placental permeability of lactate was evaluated to be about 1.3 ml/h/g of placental tissue. These results indicate that fetal concentration changes of lactate and bicarbonate due to placental transfer occur with a half time of about 4 h. It is concluded that the lactate and bicarbonate permeability of the syndesmochorial placenta of the sheep is about 20 times smaller than the placental permeability of the hemochorial placenta of the guinea pig. It seems unjustified to draw any conclusions from experimental data obtained in the sheep placenta for the transplacental acid-base balance between mother and fetus in human beings.

Evidence for pili-mediated adherence of Klebsiella pneumoniae to rat bladder epithelial cells in vitro.

The possible role of pili in the pathogenesis of urinary tract infections caused by Klebsiella pneumoniae was studied in an in vitro mixture of a phosphate-buffered saline suspension of rat bladder epithelial cells and phosphate-buffered saline-washed K. pneumoniae. Nonpiliated and piliated populations derived from a single K. pneumoniae strain were obtained by controlling the total time of growth in broth medium. The piliated phase demonstrated a significant increase in adherence when compared to the nonpiliated phase. Incubation of the bacteria and epithelial cell mixture at 4 and 37 degrees C resulted in no differences in adherence; optimal adherence occurred at pH 5. Pretreatment of the bacteria with enzymes to destroy the pili resulted in a decrease in adherence, as did killing the bacteria by various means before adherence testing. Pretreatment of the epithelial cells with certain saccharides inhibited bacterial adherence. Finally, a 96% decrease in adherence was observed after coincubation of bacteria and epithelial cells with papain-treated antipili antibodies. Thus, it appears that pili on the surface of K. pneumoniae mediate attachment of the bacteria to rat bladder epithelial cells.

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.

A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis.

Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.

Nonspecific suppressor cells in patients with chronic graft-vs-host disease after marrow grafting.

Forty-four human long-term survivors after marrow transplantation for aplastic anemia or hematologic malignancy were studied for the presence of circulating nonspecific suppressor cells. Twenty-two of the patients were healthy and 22 had mild to moderately severe chronic graft-vs-host disease (GVHD). Patient mononuclear cells (of donor origin) were tested for their ability to suppress the responses of lymphocytes obtained from the respective marrow donors to alloantigens in mixed leukocyte culture (MLC) and/or to concanavalin A (Con A). Tests were carried out between 199 and 2393 (median 376) days after transplantation. Cells from only 1 of 22 patients without chronic GVHD showed suppression of donor cell blastogeneis responses. In contrast, cells from 11 of 22 patients with chronic GVHD showed more than 30% suppression of donor cell responses in MLC and/or to Con A. The finding of suppressor cells was not related to the time of testing after grafting nor to immmunosuppressive therapy. Nonspecific suppressor activity was abrogated by irradiation with 1600 rads in vitro in five of six cases tested. Nonspecific suppressor cells may be one explanation for the severe combined immunodeficiency and the recurrent infectious complications characteristic of patients with chronic GVHD.

Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.

Long-chain alcohols are synthesized in the mouse preputial gland tumor (ESR-586) by NADPH:acyl-CoA oxidoreductase. In this study, a series of labeled acids was tested as substrates for the oxidoreductase in a cell-free system from the tumor, and the distribution of label into alcohols, waxes, and other products was determined. The system contained the labeled acid, an acyl-CoA-generating system, an NADPH-generating system, and tumor homogenate. The highest rates of alcohol synthesis were obtained with palmitic (16:0), heptadecanoic (17:0), stearic (18:0), myristic (14:0), elaidic (18:1 trans), and linoleic (18:2) acids, which yielded, respectively, 151, 124, 102, 76, 65, and 35 pmol alcohol/min per mg protein. Decanoic (10:0), lauric (12:0), oleic (18:1 cis), linolenic (18:3), arachidonic (20:4), and behenic (22:0) acids all gave lower activities. Acyl-CoA formation did not appear to be rate limiting with any of the substrates tested except behenic acid. In addition to the fatty alcohol product, a small amount of fatty aldehyde was formed in the system. Incorporation of the labeled fatty acids into wax esters was examined and the distribution of label between the alcohol and acid components of the waxes was determined. Incubation of [1-(14)C]palmitic acid yielded 3.4% free alcohol, 8.3% alcohol esterified in waxes, and 7.7% palmitoyl groups esterified into waxes, whereas, at the other extreme, [1-(14)C]linolenic acid yielded 0.8%, 0.6%, and 38%, respectively, into the homologous components.-Wykle, R. L., B. Malone, and F. Snyder. Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.

On the possible mode of action of serotonin in neurotransmission and brain dysfunction.

Criteria necessary to classify a substance as neurotransmitter are used to determine whether disturbances in neurotransmitter function are involved in brain disorders. Six rather stringent criteria for a neurotransmitter are satisfied by the biogenic amine 5-hydroxytryptamine (5-HT): 1. the availability of 5-HT within the CNS is proved by its synthesis and transport, 2. it is stored within the presynaptic bouton, 3. presynaptic stimulation releases 5-HT, 4. it reacts with receptors of the synaptic membranes. 5. a functional equivalence of presynaptic stimulation and subsynaptic 5-HT action can be demonstrated, 6. the transmission is terminated by a retrieval mechanism.--On the basis of this transmitter concept brain dysfunctions as described in the literature are discussed in the light of results obtained using in vitro techniques with isolated synaptic structures. In renal and hepatic insufficiency the transport of the 5-HT precursor tryptophan is affected at the site of the blood-brain barrier. Tryptophan-5-hydroxylase, the rate-limiting enzyme in 5-HT synthesis, is inhibited in hypoxia. The antiparkinson drug 1-aminoadamantane and its 3.5-dimethyl derivative, D 145, inhibit 5-HT catabolism as well as 5-HT storage in synaptic vesicles and reuptake into isolated nerve endings. Since 1-aminoadamantanes enhance the electrically stimulated 5-HT and DA release from nerve endings spatial shifts of transmitter substances within the synaptic area as well as direct receptor stimulation by the drugs may be the cause for the ameliorating effect.

When--and why--should nutritional state control neurotransmitter synthesis?

The rates at which neurons synthesize such Group I neurotransmitters as serotonin, acetylcholine, and the catecholamines norepinephrine and dopamine depend physiologicall on the availability to them of the circulating precursors for these compounds (tryptophan, choline and tyrosine, respectively). The concentrations of precursor in the circulation and in neurons change rapidly after food consumption, depending upon what is eaten. Nutrient intake thus normally influences the synthesis of these neurotransmitters. Neurons that emit signals by releasing serotonin, acetylcholine, dopamine, or norepinephrine participate in the control of a number of bodily functions and behaviors (e.g., hunger, food choice, sleep, alertness, sensitivity to environmental stimuli and disease states). Dietary manipulations (or the consumption of individual nutrients) can thus be used as tools for the experimental analysis of functions mediated by monoaminergic or cholinergic neurons, and as adjuncts in the treatment of some diseases of these neurons. It is unclear "why" the evolutionary process should have "allowed" the neurotransmission mediated by acetylcholine or the monoamine transmitters to be influenced by the vagaries of food choice. One possible benefit that might accrue to the organism as a result of this dependency would be the use of cholinergic or monoaminergic neurons as "sensors", providing the brain with information about peripheral metabolic state. Thus carbohydrate consumption, which--by altering plasma amino acid levels accelerates brain serotonin synthesis--enhances the release of a transmitter (serotonin) that tends to diminish the animal's desire to consume carbohydrates.

Polymicrobial bacteremia.

Of 26,961 blood cultures taken during an 18-month period at the Cincinnati General Hospital, 1,715 (6%) were positive. Ninety-four patients had blood cultures containing more than one organism. Although aerobic and anaerobic streptococci were the most frequently isolated bacteria, a variety of microorganisms, including Staphylococcus aureus and the Klebsiella-Enterobacter-Serratia group, was isolated in different combinations depending on the underlying disease. Neurological illness, malignant neoplasms, burns, and decubitus ulcers were among the most common underlying conditions found. The overall mortality was 54%, but only 58% of these deaths were specifically related to an episode of polymicrobial bacteremia. Patient survival was significantly related to appropriate antimicrobial therapy.

Peripheral chemoreceptors and exercise hyperpnea.

The carotid bodies appear to be the only peripheral chemoreceptors mediating ventilatory control during exercise in man. While little is known about the mechanism of stimulation, much is known about the effects of carotid body stimulation upon pulmonary ventilation (VE). These effects have been produced by hypercapnia, hypoxia, metabolic acidosis, arterial blood pressure, temperature, and catecholamines. A signal from CO2 flow is attractive because of the strong correlation between CO2 output and VE during exercise. The carotid body's role in the hyperpnea depends on the intensity of exercise. During heavy exercise, pH falls and hyperventilation ensues. The carotid bodies appear to be the exclusive mediators of the ventilatory compensation for the acidosis at this exercise intensity. For moderate exercise, mean arterial PCO2 does not change. Therefore, how is the CO2 signal transmitted to the respiratory center? Two current theories are: (1) since arterial PCO2 and pH oscillate with each breath, the amplitude and period of these oscillations may change during exercise and may be of sufficient magnitude to stimulate the carotid bodies, and (2) there exists a disequilibrium between hydrogen ion activity within the red blood cell and in the plasma because carbonic anhydrase is found in the former but not the latter. This theory assumes that the enzyme is not accessible to the plasma.

Glucocorticoid induction of tyrosine aminotransferase in cultured cells.

For over a decade, tyrosine aminotransferase induction in tissue culture cells has been a useful model system in which to study glucocorticosteroid action. In the 1960s, the establishment in culture of rat hepatomas expressing the inducible enzyme, already known to be induced in liver in vivo, provoked a wide-ranging series of experiments. The data from these experiments have provided considerable information regarding the mechanism of action of steroids. These include the fundamental facts that the steroids act directly on the induced cell in unmetablized form, that removal of steroid results in deinduction, that induction does not require DNA synthesis or massive changes in RNA synthesis, and that cytoplasmic receptor occupancy by active steroids correlates closely with the steroids' ability to affect inductions. Studies in tissue culture cells have led to the analysis of transcriptional and posttranscriptional models attempting to explain enzyme induction. The effects on enzyme induction of nonsteroid hormones and other factors have been studied through the use of tissue culture cells. Finally, cells and clones of cell variants are being used to study enzyme induction, through biochemical analysis and cell genetic approaches, including somatic cell hybridization.

The role of glucocorticoid hormones as biological amplifiers.

Recent research in hormone action has been aimed at studying single effects in well-defined systems. As exemplified in several chapters of this book, it has been possible to deduce a general mechanism of action of the glucocorticoids using this approach. Most hormones, and the glucocorticoids in particular, do not act as independent agents in the intact animal. Although the best known example of how glucocorticoids interact with other hormones is the amplification of the effect of those whose action is mediated by cAMP, these steroids also augment the effects of a variety of other hormones and effectors. Such interactions are of interest in clinical medicine as well, since glucocorticoid hormones are used in combination with other drugs in a number of conditions, including the treatment of asthma, allergies, and certain kinds of shock and cancer. Neither the biochemical nor the pharmacologic basis for the effects of the glucocorticoids is known. In some cases the actions of other hormones are not observed unless the tissue has first been exposed to glucocorticoids. In these instances the glucocorticoids are said to exert a "permissive effect," since they allow a process to proceed at a maximal rate even though the steroid itself has no effect on this process. There is no doubt that such examples exist, as documented above: thus the concept of a "permissive effect" does have utility. The term fails to describe the more general role the glucocorticoids play, since in many instances the steroid also has a direct effect on the process itself, or optimizes a process in which the primary effector is not as yet known. Because of these cases, and because the historically more general usage first proposed by INGLE [1] seems to have been forgotten, use of the term "permissive effect" has been avoided in this chapter. An ultimate goal in glucocorticoid hormone research is to identify the mechanisms involved in the amplification effect these hormones exert. Now that the actions of these hormones and of the hormones they interact with are being defined, such work is within the realm of feasibility.

Abnormalities of neurotransmitter enzymes in Huntington's chorea.

The activities of L-glutamate decarboxylase (GAD), GABA-transaminase (GABA-T), choline acetyltransferase (CAT), and cysteic and cysteinesulfinic acids decarboxylase (CAD/CSAD) in putamen and frontal cortex in both Huntington's chorea and normal tissues were measured. The greatest difference between Huntington's and normal tissues occurred in putamen, in which the apparent CSAD activity was reduced by 85%, while no difference was observed in frontal cortex. GAD, CAD, and CAT activities were also reduced in putamen by 65%, 63%, and 42%, respectively (P less than 0.05). Slight reduction in the enzyme activities was also observed in frontal cortex. However, these reductions appeared to be statistically insignificant (P greater than 0.05 in all cases). GABA-T showed little difference in both putamen and frontal cortex in Huntington's chorea and normal tissues. GAD and GABA-T from Huntington's tissues were indistinguishable from those obtained from normal tissues by double diffusion test and by microcomplement fixation test, which is capable of distinguishing proteins with a single amino acid substitution. Furthermore, the similarity of the complement fixation curves for GAD from Huntington's and normal tissues suggests that the decrease in GAD activity is probably due to the reduction in the number of GAD molecules, presumably through the loss of neurons, and not due to the inhibition or inactivation of GAD activity by toxic substances which might be present in Huntington's chorea.

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.

Occurrence and role of tightly bound adenine nucleotides in sarcoplasmic reticulum of rabbit skeletal muscle.

Freshly isolated sarcoplasmic reticulum vesicles contain 0.05 mol of tightly bound ADP and 0.03 mol of tightly bound ATP per mol of Ca2+, Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). These values were increased to 0.1-0.2 mol ADP and 0.2-0.3 mol ATP per mol of ATPase after incubation of vesicles in the presence of MgATP and Ca2+ at 25 degrees C and pH 7.0. Half-maximal enrichment of tightly bound nucleotides was obtained with 2.5 mM ATP and 0.32 microM free Ca2+. Uncoupling of calcium transport from ATPase activity by mild acidic conditions or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at pH 7.0 decreased the ability of the membranes to be enriched with tightly bound nucleotides and also decreased the content of tightly bound nucleotides of previously enriched membranes. Tightly bound [3H]nucleotides could only be partially displaced by reincubation under enrichment conditions. Tightly bound nucleotides are associated with energized calcium translocation but do not appear to be directly involved in the catalytic cycle.

Pollution of the sea and its effects.

Marine pollution has been studied under the following groupings of effects; harm to living resources, hazards to human health, reduction of amenities and interference with other users of the sea. This paper is concerned mainly with the first two categories and their interrelation. Apart from certain seabirds affected by oil, the major stocks of marine animals show no evidence of reduction by pollution. Pollution effects are generally insignificant in relation to other factors governing reproductive success, survival, growth and population size. Even in the North Sea, which has received a greatly increased pollution load during the last three decades, both total production of fish and catch per unit of effort (a measure of abundance) of cod, haddock and plaice increased during the 20 years 1950--69. Very recent decreases have been due to over-exploitation but, except in certain estuaries and immediate coastal waters, direct damage by pollution to marine populations and ecosystems is not evident. Pollution effects can, however, be detected by chemical analysis. The paper examines human health risks arising in the marine environment, particularly from contaminated seafood, especially in relation to sewage pollution, metals such as mercury, cadmium and lead, synthetic organic substances and oil.

The pattern of disease in the post-infection era: national trends.

Information that can be used to assess trends in the health of the population is limited to the results of irregular surveys of nutritional status and 'I.Q.', to data obtained from the notification of infectious diseases, congenital malformations, blindness and other selected defects, and to mortality rates. The last have been recorded since 1841 and provide the most detailed and useful information, although they are often difficult to interpret because of changes in the nomenclature, classification, methods of diagnosis, and efficacy of treatment of disease states. In the last 40 years, mortality rates have shown progressive reductions at all ages which have continued past the time when improvements in the prevention and treatment of infectious disease might be expected to have produced their principal benefits. Notable differences have emerged between the sexes, the rates continuing to decline in women but remaining more or less stable for a period in middle-aged men. This difference can be attributed to sex differences in life-style, so that until recently the trends in women are likely to have been the better indicators of the effect of toxic agents in the environment. The available data are inadequate to assess possible effects such as alterations in behaviour, but are of some help in regard to teratogenicity and carcinogenicity.

Molecular mechanisms regulating the interactions between the benzodazepines and GABA receptors in the central nervous system.

Using radioreceptor assay techniques to measure the kinetics of GABA and diazepam receptors, a relationship between GABA and benzodiazepine receptors has been firmly established in membranes of brain and neuroblastoma NB2a clonal cell lines. Occupancy of benzodiazepine receptors uncovers a new population of GABA receptors (GABA2 receptors) endowed with high affinity for GABA. Moreover, stimulation of GABA receptors increases the affinity of 1,4-benzodiazepine receptors for 1,4-benzodiazepines. This reciprocal interaction appears to be mediated by an endogenous regulatory protein (for details on this protein see [14 and 29]) which allosterically regulates GABA2 receptors while it competitively interacts with benzodiazepines for their specific binding sites. The rank order of potency of the various 1,4-benzodiazepines to block the action of this protein inhibitor on GABA receptors is related to their capacity to displace 3H-diazepam binding. These data suggest that the interaction between the 1,4-benzodiazepine receptors and the endogenous protein modulator of GABA2 receptors might play a role in the pharmacological action of the 1,4-benzodiazepines.

Physico-chemical limitations in experimental investigations.

An element or compound in a natural water system is usually distributed between a variety of physico-chemical forms, both dissolved and particulate. The distribution is determined by the properties of the ion or molecule in question and by a number of major variables, including ionic strength, the nature and concentrations of major dissolved elements, particulate matter and organic complexing material, pH and the electron activity (pE); it may thus vary widely between different environments. The design of experiments to study sublethal effects of pollutants in sea water ideally requires that the test medium is closely matched to the environment for which information is needed, with respect to the ranges of concentration and activity, and the chemical speciation, of the pollutant and of any other constituents which may influence its effects. This in turn requires either that the pollutant can be added in the appropriate forms, implying a knowledge of the existing speciation, or that the added material rapidly exchanges with the forms already present. The implications of these requirements are most apparent for those pollutants that show complex chemical behaviour in sea water. This account concentrates on metals of toxicological significance. Consideration of particulate associations, redox speciation, and complex formation in the dissolved state with inorganic and organic ligands, suggests that physicochemical factors limit the usefulness, in terms of environmental predictions, of experimental studies of biological effects of metals, both inherently and through inadequate knowledge of environmental speciation and the mechanisms and rates of interconversion between species. Of particular importance are non-equilibrium features in speciation, such as the presence of thermodynamically unstable oxidation states and of kinetically non-labile associations. Interpretation of the nature of these associations is complicated by the presence of colloidal and organic macromolecular material in dissolved fractions as conventionally defined. While the chemical behaviour of some substances in sea water is considerably less complicated than that of the trace metals, there is a need with all types of pollutants for greater attention to physico-chemical factors in both the design and interpretation of experiments to investigate biological effects.

Measurement of the responses of individuals to environmental stress and pollution: studies with bivalve molluscs.

Certain physiological differences between individuals in different populations of the mussel, Mytilus edulis, are described. In particular, the scope for growth differs in space and time and may be used to assess the animals' physiological condition. When the required measurements are made in the field, the rates of growth predicted from the physiological data agree well with observed rates of growth. An alternative approach utilizes mussels transplanted to various waters, with indices of condition then measured in then measured in the laboratory under standard conditions; an example of this approach is illustrated. Laboratory experiments are used to equate various levels of physiological condition with fecundity, in an attempt to equate physiological effects on the individual with likely population damage. A cytochemical index of stress is described, based on the latency of lysosomal enzymes; spatial variability in this index, and its relation with the scope for growth, are discussed. Finally, the results of some experiments on the effects of petroleum hydrocarbons on mussels are described and the presence of inducible activity of NADPH-dependent tetrazolium reductase in the blood cells is demonstrated. Certain considerations that apply in adopting similar measurements of biological effects of pollution in environmental monitoring programmes are discussed.

[Relationship between the presence of meconium in newborn lambs and postnatal pH and blood gas tension levels (author's transl)].

The pH of the blood and blood gas tension (PO2 and PCO2) were estimated in thirty-two lambs. Blood was taken from the umbilical vein during labour and blood was collected from the left ventricle after birth (1, 10 and 60 minutes). The findings in twenty-one lambds were used to determine the relationship between the presence of meconium on the fleece and the postnatal changes in pH and blood gas tension. pH, blood gas tension and vitality were found to be identical both in the meconium-stained and in the unstained lambs. Within this experiment, there was no relationship between the length of parturition and pH and blood gas tension levels. The pH, PO2 and PCO2 as determined in the umbilical blood showed that the lambs (both meconium-stained and unstained) continue to be in good condition throughout a normal parturition. Postnatal adjustment is rapid and, following normal ventilation, pH, PO2 and PCO2 respectively attain levels of 7.379; 54.7 and 43.7 within sixty minutes. Diminished vitality, resulting from pathological factors, was observed in four lambs.

Polymorphism of the second component of human complement (C2). Observation of the rare phenotype (C2 2 (= C2 B) and data on the localization of the C2 locus in the HLA region.

The polymorphism of the second component of human complement was studied by means of isoelectric focusing in polyacrylamide gels with subsequent complement-dependent lysis of sensitized sheep erythrocytes in an agarose overlay containing C2-deficient or normal human serum. In a material of 289 unrelated individuals the following gene frequencies were observed: C21=0.965 and C22=0.035. The rare phenotype C2 2 (=C2 B) could be seen once in a child of a C2 1--2 heterozygous mother. The investigation of the C2/HLA relationship revealed a very close linkage: Among 62 informative meiotic divisions one recombination between HLA-B and C2 was found (i.e. 1.61%); in addition, C2(2) was significantly associated with HLA-B15 and -Cw3. In a family with an HLA-B/D(DR) crossover C2 segregated together with HLA-D(DR). This supports the assumption of a C2 structural locus outside HLA-B, probably near HLA-D(DR).

[The effects of Pavulon (pancuronium bromide) on maternal circulation and metabolism as well as on fetal metabolism and postnatal condition at Caesarean section (author's transl)].

The effects of pancuronium bromide (Pavulon) on maternal circulation and metabolism as well as on fetal metabolism and postnatal conditions were studied in 13 women in late pregnancy (between gestational weeks 33 and 40) requiring Caesarean section in general anaesthesia. A comparison with results of 50 spontaneous deliveries without analgetics was performed. The average dosage of 4 mg did not effect the maternal blood pressure, fetal muscle tonus and cardiorespiratory adaption of the newborn. According to general anaesthesia we found in acid-base status a pH decrease with respiratory acidosis in comparison with spontaneous deliveries without anaesthetics, but pH and pCO2 were normalized 30 min after delivery. On the contrary, the oxygen tension were higher as at delivery as 30 min after delivery by Caesarean section. Although 1 minute Apgar score was lower than after spontaneous delivery the heart rate and frequency of ventilation were normal in every time and in the following minutes Apgar score was 7 to 10, too. An effect of pancuronium bromide to maternal or fetal carbohydrate metabolism could not be found. The less side effects on maternal cardiovascular system and the missing influence in postnatal condition recommand pancuronium bromide as a suitable relaxant in obstetrical anaesthesia, too.

[Regulation processes in biological systems. II. Role of compartmentation and of organized multienzyme systems].

The picture, emerging from the experimental results on the physical association of multienzyme systems, is that the true physiological significance of the aggregated state can be understood only if it is correlated to the structural and functional integration of the cellular metabolic framework as a whole. The enzyme clusters exhibit two distinct functional properties. The first is the spatial translocation of intermediate substrates, the effect of which may be viewed as metabolic "channelling" or "vectorial catalysis" if the enzyme clusters are arrayed in some manner in the cell. The second is the coordinate regulation which represents an efficient and economical mean of controlling two or more functionally related enzymes. The common element to these two properties is the spatial character, which is potentially present in the function and the regulation of the intermediary metabolism. The biological systems, and metabolism in particular, exhibit both stability and variability; the latter sometimes assumes the character of periodicity. Whether the oscillations have a definite importance at the level of the intermediary metabolism itself, may well be questioned; the oscillatory faculties may rather serve as elements to be used in more complex functions of the biological systems. A thorough understanding of the role of clustered multienzyme systems and of the oscillatory phenomena in cellular metabolism demands a clearer physicochemical picture of the dynamic state of the living cell than we have at present. For this reason some of the generalizations derived from in vitro studies of single, isolated enzyme activities are not justified.

Immunoblastic lymphadenopathy, systemic lupus erythematosus, and related disorders. Possible pathogenetic pathways.

The authors discuss the hypothesis that the spontaneously developing (angio-) immunoblastic lymphadenopathy of man as well as the various autoantibodies and constitutional symptoms accompanying this disease may be mediated by different reactions of T lymphocytes toward adjacent lymphocytes and macrophages, whose membranes were rendered incompatible by certain viruses or sensitizing drugs such as the antiepileptic compound diphenylhydantoin. This concept is based on two different lines of experimental evidence: (1) results obtained with animal graft-versus-host reactions, in which immunoblastic lymphadenopathy, angiogenesis, dermatitis and multiple autoantibody formation are known to be induced by reactions of parental T lymphocytes toward genetically foreign structures of the major histocompatibility complex; (2) experiments pointing to an essential similarity in T-cell reactions toward genetically foreign major histocompatibility structures on the one hand and self-major histocompatibility structures that were rendered "foreign" by viruses or chemicals on the other hand; (3) recent findings in mice that demonstrate a T-cell-dependent lymphoproliferation after the administration of diphenylhydantoin.

[Therapy groups with patients and nursing staff in a geriatric hospital (author's transl)].

Within a very short time the non-demented, chronically hospitalized old person turns into a hospital-activity-oriented patient. He thereby gives up a considerablle part of his personality. In order to understand this process, special attention has to be paid to the intertwining of the dynamics of the chronic geriatric patient with the dynamics of the nursing staff. As a therapy experiment we have been conducting parallel talk-groups with patients and with their nursing staff for one year. As a result, the patients showed increased activity, increased dynamism, improved contact ability, increased interest in the outside world, and improved affect. The nursing staff also reported improvement of the group-patients' condition on the floor. With the nursing staff we found a reduction of the defensive mechanisms, a separation from old nursing-role definitions, acceptance and encouragement of the patients in their new role, and improved satisfaction with patient contact. On the other hand, the nursing staff expects more personal contact to come from the patients. The groups have shown to be useful and have to a large extent reached the therapy goals.

[Clinical test of the Cerebral Function Monitor in intensive care].

Due to its simplicity, and because of the lack of an instrument designed for the routine monitoring of cerebral function in intensive care, the M.C.F. was used on patients presenting with disorders of consciousness resulting from various causes, including: exogenous poisoning; cranial trauma; cerebral anoxia. 15 examples are thus presented to illustrate the different clinical states. This apparatus revealed itself to be of particular use in the instantaneous evaluation of a patient's overall cerebral activity, as well as in the assessment of the patient's clinical prognosis. The slow speed of the trace enabled us to make continual recordings lasting several weeks. It was thus possible to compare immediately the levels of the disease. Furthermore, all variations--even of a transitory nature--of the level and fluctuations of this activity could be detected at all times. The M.C.F., therefore, would seem to be a very useful aid--perhaps indispensable--as a monitor of the comatose patient in an Intensive Care Unit.

Beta-1-3-glucanase and dimorphism in Paracoccidioides brasiliensis.

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of beta-glucanases, low amounts of alpha-glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of beta-1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of beta-1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60 degrees C and pH of 5.0 (acetate buffer or pH 6.0 (phosphate buffer). Its Km was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and Km were also slightly different. Following treatment of the cell walls with chitinase, beta-1,3-glucanase was released into the medium.

Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro.

Experiments with isolated pancreatic islets or dispersed islet cells from non-inbred ob/ob mice were performed to test the hypothesis that free radicals, notably OH., mediate the diabetogenic toxicity of alloxan. Accumulation of 86Rb+ by whole islets and exclusion of Trypan Blue by dispersed cells were used as previously validated criteria of islet-cell viability. Alloxan alone drastically inhibited the Rb+ accumulation and significantly decreased the frequency of cells excluding Trypan Blue. Enzymic scavengers of O2.- and H2O2 or non-enzymic scavengers of OH. or singlet oxygen were added to the incubation medium and tested for their ability to protect against these effects of alloxan. Superoxide dismutase, catalase, dimethyl sulphoxide, benzoate, and mannitol counteracted the effects of alloxan in both cytotoxicity assays. Significant protection of the Rb+-accumulating capacity was also afforded by butanol, caffeine, theophylline, NADH, NADPH and, to a small extent, NAD+. Urea has a poor affinity for OH. and did not protect against alloxan. No effect was obtained with the singlet-oxygen scavenger, histidine. Except for the protection by NADH and NADPH, which may be due to a direct reaction with alloxan in the medium, the results strongly support the hypothesis. beta-Cells may be particularly vulnerable to alloxan because their metabolic specialization facilitates reduction of the drug and perhaps of other substrates for O2.--yielding redox cycles.

Amoxapine in experimental psychopharmacology: a neuroleptic or an antidepressant?

2-Chloro-11-(piperazinyl)dibenz[b,f][1,4]-oxazepine (amoxapine) gives an unusual spectrum in psychopharmacological tests. Many of its effects are similar to those of neuroleptics: sedation, decrease in motor activity, catalepsy (which is, however, qualitatively different from that induced by classical neuroleptics), transitory suppression of avoidance reaction, antagonism of amphetamine induced toxicity in crowded mice and inhibition of stereotyped behavior induced by amphetamine in rats, and antagonism to various effects of apomorphine (stereotyped behaviour in rats, climbing behaviour, stereotyped behaviour and hypothermia in mice). At similar doses which produce the above mentioned effects, amoxapine also shows effects atypical for a neuroleptic, but which are relatively characteristic of antidepressants: antagonism of prochlorperazine-induced catalepsy in rats, inhibition of reserpine induced hypothermia in mice and enhancement of yohimbine toxicity in mice. The profile of this substance does not facilitate the anticipation of therapeutic effects in humans.

Proestrus and metestrus rat uterus, a rapid and simple in vitro method for detecting histamine H2-receptor antagonism.

The antagonism caused by metiamide and cimetidine on histamine-induced inhibition of spontaneous and electrically stimulated isometric contractions of superfused rat uterine horns from proestrus and metestrus stages was studied in vitro. Histamine depressed smooth muscle "twitch" responses of spontaneously contracting or electrically stimulated uterine preparations of both stages in the same dose-dependent manner. The typical effects of organ relaxation, inhibition of contraction height and reduction of resting tension generated by histamine, could both be antagonized by the histamine H2-receptor blockers metiamide and cimetidine, while both compounds failed to reverse orciprenaline- or isoproterenol-induced inhibition of uterine contractility. Diphenhydramine, a histamine H1-receptor antagonist, was not able to reduce histamine-induced inhibition of uterine contractions, thereby confirming that the histamine receptors of the rat uterine tissue are H2 in type. Furthermore, beta-adrenergic blockers, like propranolol and dichloroisoproterenol, failed to antagonize the decrease in contraction amplitude but prevented fall in resting tension induced by histamine. Tyramine, cAMP or dibutyryl-cAMP produced no inhibition of motility of the isolated uterine tissue. Possible mechanisms of these findings are discussed. The findings show that the isolated non-estrus rat uterus is useful as a rapid method for investigating specific effects of drugs designated for potential histamine H2-receptor antagonism. This preparation offers the additional advantage that no interference with beta-blockers occurs.

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.

Purification and properties of a proteinaceous metallo-proteinase inhibitor from Streptomyces nigrescens TK-23.

A novel metallo-proteinase inhibitor which is capable of inhibiting the activities of metallo-proteinases such as the thermolysin, was isolated from the culture filtrates of Streptomyces nigrescens TK-23. The inhibitor was purified batch-wise from the culture filtrate by Amberlite IRC-50 and column chromatographies on CM-Sephadex C-50 and Sephadex G-50. The purified inhibitor showed a single band on 15% polyacrylamide gel electrophoresis at pH 4.3, and at pH 7.5 on SDS-gels. The inhibitor retained 80% of its original activity after treatment of 100 degrees C for 5 min between pH and 7. The molecular weight was estimated to be 12 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, and calcuated as 11 950 from its amino acid composition. The isoelectric point was pH 10.3. The inhibitor showed a high content of hydrophobic amino acids, did not contain tryptophan, and had two disulfide bridges. It also showed specific inhibitory activity for metallo-proteinases but not for serine-, thio- and carboxyl-proteinases.

Alpha 2-adrenoceptors mediate clonidine-induced sedation in the rat.

1 The central alpha-adrenoceptors responsible for mediating clonidine-induced sedation in rats have been characterized according to their sensitivity to alpha-adrenoceptor agonists and antagonists.2 Clonidine, injected intraperitoneally or intracerebroventricularly, caused dose-dependent sedation, both in terms of a reduction in the time that rats could remain on an accelerating rotarod and in terms of overt sedation assessed visually. Following intracerebroventricular injection, xylazine, naphazoline and methoxamine, but not phenylephrine, produced similar effects.3 The sedation caused by intraperitoneal injection of clonidine was antagonized by intracerebroventricularly injected phentolamine, yohimbine, piperoxan and tolazoline but not by labetalol, thymoxamine or prazosin.4 The relative potencies of the agonists in causing sedation and of the antagonists in inhibiting the sedative effect of clonidine clearly demonstrated that the central alpha-adrenoceptors mediating clonidine-induced sedation are the same as the peripheral presynaptic alpha(2)-adrenoceptors.5 All the alpha-adrenoceptor agonists caused hypothermia after intracerebroventricular injection, but their order of potency was different from that in producing sedation. The hypothermic effect of intraperitoneally injected clonidine was little affected by any of the antagonists administered intracerebroventricularly. No conclusions could be drawn concerning the type of receptor responsible for mediating hypothermia.

The effects of high pressure helium and nitrogen on the release of acetylcholine from the guinea-pig ileum.

1 The effects of high pressures of helium and of nitrogen on acetylcholine release were tested using the guinea-pig ileum as a model preparation. A superfusion system was designed in which this tissue could be maintained under physiological conditions in a high pressure chamber.2 Helium, at a pressure of 136 atm slightly increased the spontaneous output of acetylcholine but produced no significant changes at 68 atm (136 atm is close to the lethal pressure for small mammals).3 The acetylcholine release evoked by electrical stimulation or by 55 mM potassium was not altered by 136 atm of helium. Effects on tetrodotoxin-treated tissues were not consistent.4 Nitrogen, which in contrast to helium possesses general anaesthetic properties, caused considerable increases in spontaneous and in electrically evoked acetylcholine output at pressures which produce anaesthesia. These increases were not changed when helium was used to increase the total pressure to 136 atm, although this reverses the general anaesthetic actions of nitrogen in vivo.5 The increases in rate of acetylcholine release produced by nitrogen were observed in tetrodotoxintreated tissues and in tissues from reserpine-treated animals. In a calcium-free medium the increases were considerably smaller.6 The conclusions from these results are that while high pressures of helium caused little or no change in acetylcholine release rates, nitrogen produced large changes, which were not due to effects on axonal conduction. The effect of nitrogen is not apparently related to its general anaesthetic actions. Differences such as these in transmitter release would be likely to contribute to the differing physiological effects of these two gases.

Nerve growth factor: effects on D-amphetamine-induced activity and brain monoamines.

Adult male rats were given 6-hydroxydopamine lesions of the nucleus accumbens, followed immediately by injections of saline or nerve growth factor (NGF; 125 B.U.) near the substantia nigra. Such lesions were previously reported to attenuate the locomotor response to D-amphetamine. NGF-treated rats showed an enhanced response to D-amphetamine (1.5 mg/kg) when tested 15 days postoperatively. Levels of dopamine and norepinephrine in the striatum and nucleus accumbens were equivalently depressed in the two lesion groups, indicating that the apparent recovery of the NGF-treated rats was probably not due to catecholaminergic neuronal regrowth. Intracerebral NGF administeration enhanced the response to D-amphetamine 15 days later in rats without lesions, and also appeared to result in increased turnover of brain norepinephrine and serotonin at 3, but not 15, days postadministration. NGF might increase dopamine turnover at 15 days, but the evidence obtained did not convincingly confirm or negate this possibility. The results in brain-damaged and intact rats, and also modify the apparent turnover of brain monoamines.

Binding characteristics of a major protein in rat ventral prostate cytosol that interacts with estramustine, a nitrogen mustard derivative of 17 beta-estradiol.

The tissue distribution of [3H]estramustine, the dephosphorylated metabolite of estramustine phosphate (Estracyt), in the male rat was compared to that of [3H]estradiol 30 min and 2 hr following i.p. administration. In contrast to estradiol, estramustine was found to be efficiently concentrated in the ventral prostate gland by a soluble protein. The binding characteristics of this protein were studied in vitro using cytosol preparations of the gland. With a dextran-coated charcoal technique, the protein was found to bind estramustine with a broad pH optimum between pH 7 and pH 8.5, with an apparent Kd of 10 to 30 nM, and with a binding capacity of about 5 nmol/mg cytosol protein. The estramustine/protein complex was not retained by DNA-cellulose. None of the natural steroids tested inhibited the binding of 10 nM [3H]estramustine by more than 35% (progesterone), even when added in 4500-fold excess. The presence of a nitrogen mustard moiety at position 3 of the steroid was necessary for high-affinity binding to the protein. The protein was calculated to constitute about 20% of the total cytosol protein content.

Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties.

A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine NaOH was used as a buffer. The apparent Michaelis-Menten (KM) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 mumol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl groups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +/- 3.22 units per mg protein (mean +/- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mextures of RBC membranes with relatively "low" and relatively "high" activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors.

Strain differences in adrenal xenobiotic metabolism in guinea pigs.

Studies were carried out to compare adrenal and hepatic xenobiotic metabolism in various strains of guinea pigs. In all strains studied (Hartley, English Short Hair, NIH, Strain 2, Strain 13), microsomal protein and cytochrome P-450 levels and NADPH-cytochrome c reductase activities were greater in adrenals than livers. Neither adrenal values nor hepatic values for these parameters differed across strains. Ethylmorphine (EM) demethylase and benzo[a]pyrene (BP) hydroxylase activities were also greater in adrenals than livers in all strains. However, the rates of adrenal xenobiotic metabolism were far greater in the highly inbred Strain 2 and Strain 13 guinea pigs than in other strains. In contrast, hepatic metabolism of EM and BP was not strain-dependent. Adrenal steroid 21-hydroxylase activity was also similar in all strains. The results indicate that strain is an important determinant of adrenal but not hepatic xenobiotic metabolism in the guinea pig. In addition, genetic control of adrenal microsomal drug and steroid metabolism appear to be independent of one another.

Aldrin epoxidation, a highly sensitive indicator specific for cytochrome P-450-dependent mono-oxygenase activities.

Aldrin epoxidation was studied in rat liver microsomes. The assay is very sensitive; amounts of less than 1 microgram of microsomal protein were sufficient for activity determination. The very low background, stability of the metabolite, and the complete separation of substrate and metabolite permit estimation of mono-oxygenase activities of less than 1 pmol per mg of protein per min by a simple procedure. Pretreatment of animals with the mono-oxygenase inducer phenobarbital (PB) increased the epoxidation rate 3-fold, whereas 3-methylcholanthrene (MC) treatment markedly depressed enzyme activity. Induction with MC did not change the apparent Km of the reaction, which was 18 muM. The Km in microsomes from PB-treated animals was 28 muM. These data suggest that the same or (a) similar form(s) of mono-oxygenase catalyze(s) the epoxidation in the three different microsomal preparations. SKF 525-A, metyrapone, and 7,8-benzoflavone were almost similarly active as inhibitors in microsomes from control and inducer-treated rats. Sensitivity to these inhibitors was low; 0.7 mM SKF 525-A and 0.4 mM 7,8-benzoflavone were necessary to reduce enzyme activity by 50%, whereas 0.5 mM metyrapone caused an inhibition of 10-45%. The activity of aldrin epoxidation in untreated rats increased almost parallel to the activity of ethylmorphine demethylation between 3 and 10 weeks of age. The rate of benzo[a]pyrene hydroxylation remained unchanged during this period. The results demonstrate that aldrin epoxidation offers a selective and sensitive assay for the activity of mono-oxygenases dependent on cytochrome P-450 forms.

A neurophysiological analysis of the effects of hypercapnia on the embryonic spinal cord.

Progressive hypercapnia in the normal chick embryo late in incubation (14-19 days) is temporally associated with a gradual decline in motor activity and the corresponding frequency of polyneuronal (burst) activity in the spinal cord. We have studied the possible correlation between the increasing hypercapnia and the declining frequency of burst activity seen during these later stages of incubation by systematic manipulation of CO2 levels. Burst frequency was seen to decrease as a result of a 5-min exposure to different carbon dioxide environments at all ages studied. The magnitude of this inhibition and the ability to recover from consecutive bouts of hypercapnia (pulses) is pulse and age dependent. These short-term (less than 5.0 min) changes differ qualitatively from the long-term (greater than 2.5 h) effects of subsequent hypercapnic episodes. This evidence suggests a role for metabolic factors in the normal developmental changes in motility and electrophysiological activity in the chicken embryo spinal cord.

Blood pressure and plasma noradrenaline during single high-dose beta adrenoceptor blockade.

The acute effects upon blood pressure and sympathetic outflow of two beta adrenoceptor blocking drugs, propranolol and atenolol, are described in five healthy normotensive subjects. Supine blood pressure, heart rate, plasma noradrenaline, and urinary catecholamine excretion were measured before and at intervals for 24 h after a single oral dose of either propranolol 200 mg, atenolol 100 mg, or placebo. Propranolol caused a fall in blood pressure and heart rate of 17.2/14.1 mm Hg and 20.4 beats/min respectively two hours after dose. Atenolol caused a fall in blood pressure of 11.4/18.6 mm Hg within 7 h of the dose, and a fall in heart rate of 13.8 beats/min after 2 h. The reduction in blood pressure after single high dose beta adrenoceptor blockade is established. The synchronous reduction in blood pressure and heart rate after propranolol was not associated with an increase in peripheral sympathietic activity as assessed by the biochemical indices. It is conceivable that the reduction in blood pressure during beta adrenoceptor blockade may be due in part to inappropriately low sympathetic activity but this cannot be the main mechanism of pressure reduction.

Functional differentiation of mouse T lymphocytes. GVHR-precursors: tissue origin and specificity of activity inducer.

For theoretical and practical reasons, it is important to find out whether the differentiation of T cell precursors to the functional lymphocytes can be induced under in vitro conditions. Using the local GVHR assay (based on the enlargement of the popliteal lymph node), the inducibility of the precursors of reactive cells was studied with bone marrow, thymus, spleen, and lymph node cell suspensions submitted to short-term incubation with cell-free extracts from calf thymus, spleen or brain. GVHR-precursors from bone marrow were inducible not only specifically (i.e., with thymus extract) but also--and even to a higher degree--with spleen or brain extract. Thymus and spleen cell suspensions (the latter also depleted of the reactive subpopulation by treatment with anti-Thy 1.2 serum and complement) were, on the other hand, inducible mainly specifically, whereas lymph node cells were refractory to induction. The inductive action of tissue extracts obviously depends on the tissue origin of T cell precursors; their effects on pre- and postthymic differentiation of T lymphocytes are discussed.

Studies on the interaction of tiamulin with the phospholipids in model membranes and with microsomes.

The drug tiamulin interacts with phospholipid membranes mainly in a nonelectrostatic way. At pH-values where the drug possesses a net positive charge only small binding is observed. In the presence of cholesterol tiamulin is excluded from the membranes. The interaction of tiamulin with membranes cannot be explained by a simple partitioning but is governed by structural rearrangements of the lipid phase. At low drug concentrations we observe sigmoidal binding characteristics in the rigid as well as in the fluid state up to a level of about 2-3 mol drug bound per 1000 mol phospholipid. The range in which this cooperative interaction occurs can be compared with the drug concentration in the erythrocyte membrane which protects from hypotonic lysis. Further addition of tiamulin to the rigid membrane leads to fluidization. Saturation of the membranes with tiamulin is completely in parallel to their fluidization. The relevance of the cooperative interaction at low drug concentration and of the subsequent fluidization at elevated concentration for the microsomal membrane is discussed.

Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae.

Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis.

Large-scale production and physicochemical characterization of human immune interferon.

Large-scale production of crude high-titered (10(2.3) to 10(4) U/ml) human immune interferon (type II) was carried out in roller bottle cultures of human peripheral lymphocytes by using the T-cell mitogen staphylococcal enterotoxin A. Over 99% of human immune interferon was destroyed by pH 2 or heat at 56 degrees C for 1 h. The interferon was not neutralized by antibody to human leukocyte interferon. The kinetics of development of the antiviral state were slow for immune interferon relative to those for leukocyte interferon. Ultrogel AcA 54 chromatography of crude or the concentrated interferon resulted in two peaks of activity, a major one (87% of recovered activity) with a molecular weight of 40,000 to 46,000 and a minor peak of molecular weight 65,000 to 70,000. The column elution buffer consisting of 18% ethylene glycol and 1 M NaCl in phosphate-buffered saline resulted in at least 100% recovery of added interferon. The data suggest, then, that the interferon produced under large-scale conditions was immune (type II). The efficiency of the production was comparable to that described for large-scale production of human leukocyte interferon. Our large-scale production system for human immune interferon offers a feasible approach to preparation of large quantities of purified immune interferon for structure studies, antibody production, and clinical application.

Attempts to induce ovulation in amenorrheic patients using D-Ala-6-LH-RH propylamide.

Thirteen amenorrheic patients, two with primary amenorrhea, and eleven with secondary amenorrhea, including five with anorexia nervosa, were treated with an analog of LH-RH, D-Ala-6-desGly-10-LH-RH propylamide (D-Ala-6-LH-RH PA). The patients were follwed with checks of daily basal body temperature, cervical mucus characteristics, urinary total estrogens and pregnanediol, plasma LH and FSH, and twice-weekly clinical checkups. D-Ala-6-LH-RH PA was given continuously for 11 days at a dose of 50 microgram im from the 4th day of a steroid-induced cycle. Ethynyl estradiol (50 microgram orally) was given twice on day 14 followed by 250 microgram of D-Ala-6-LH-RH PA for the following 3 days. All the treated cycles were monophasic. Withdrawal bleeding occured in nine patients from the 4th to 6th days posttreatment. A significant total maximal increment in estrogens was found in seven patients, which was higher than the values obtained previously in the same patients with human menopausal gonadotropin. One patient conceived in the cycle immediately following discontinuation of treatment, indicating a possible effect of analog on the follicular development during the treatment; a normal baby was delivered. A second patient conceived three cycles afterwards, and two other patients began normal menstrual cycles.

Serum progesterone and estradiol in pregnant women selected for progestagen treatment.

Patients with two or more previous spontaneous second trimester abortions and vaginal cytology indicating a poor progestational response in current pregnancies were selected for treatment with Provera (medroxyprogesterone acetate) and/or Delalutin (17 alpha-hydroxyprogesterone caproate). Serum was examined serially for progesterone (P) and estradiol (E) by radioimmunoassay. Serum from 174 untreated patients with no known complications ranging from 6--40 weeks gestation provided normal distribution data. Of 14 progestagen-treated patients, four aborted during the second trimester. These all had chronically low (greater than 50% of observations were less than 1 standard deviation of the normal population) or falling P/E ratios. The rest delivered normal full-term infants although five of the 10 had chronically low P, seven had chronically low P/E ratios, and in one other P/E was falling. Chronically high E contributed to the low P/E ratio in three cases. Thus, these selected cases with poor obstetrical histories demonstrated steroid patterns outside the +/- 1 standard deviation range, although the steroid levels were still within the normal range. Serum progesterone and estradiol analysis may eventually be useful in identifying patients who will best respond to progestagen treatment.

Sucrose transport by the Escherichia coli lactose carrier.

Several lines of evidence suggest that sucrose is transported by the lactose carrier of Escherichia coli. Entry of sucrose was monitored by an osmotic method which involves exposure of cells to a hyperosmotic solution of disaccharide (250 mM). Such cells shrink (optical density rises), and if the solute enters the cell, there is a return toward initial values (optical density falls). By this technique sucrose was found to enter cells at a rate approximately one third that of lactose. In addition, the entry of [14C]sucrose was followed by direct analysis of cell contents after separation of cells from the medium by centrifugation. Sucrose accumulated within the cell to a concentration 160% of that in the external medium. The addition of sucrose to an anaerobic suspension of cells resulted in a small alkalinization of the external medium. These data are consistent with the view that the lactose carrier can accumulate sucrose by a proton cotransport system. The carrier exhibits a very low affinity for the disaccharide (150 mM) but a moderately rapid Vmax.

Purification and properties of mouse liver fructose 1,6-bisphosphatase.

A simple procedure has been developed for the purification of mouse liver and kidney fructose-1,6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143,000, subunit size was 37,500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1,6-bisphosphate was 1.3 microM. The Ki value for AMP was 19 microM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.

Amino acid sequence of the biotinyl subunit from transcarboxylase.

The complete amino acid sequence of the biotinyl subunit from the enzyme transcarboxylase of Propionibacterium shermanii has been determined from the structures of overlapping tryptic and cyanogen bromide peptides together with sequenator analysis on the whole subunit. The subunit contains 123 amino acid residues. Eleven of nineteen residues in the region of biotin attachment, when compared to pyruvate carboxylase from avian liver (Rylatt, D. B., Keech, D. B., and Wallace, J. C. (1977) Arch. Biochem. Biophys. 183, 113-122), were found to be in identical positions relative to biocytin. There was less homology with acetyl-CoA carboxylase from Escherichia coli (Sutton, M. R., Fall, R. R., Nervi, A. M., Alberts, A. W., Vagelos, P. R., and Bradshaw, R. A. (1977) J. Biol. Chem. 252, 3934-3940), but in all of these biotin enzymes there was an alanylmethionyl-biocytinyl-methionine sequence. The secondary structure of the biotinyl subunit has been estimated using the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148) and considered in relationship to the role of the biotinyl subunit in the structure and function in transcarboxylase.

Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Because of previous data suggesting that aminoacyl-tRNA synthetases make a transient Michael adduct with a specific uridine residue in the tRNA structure, (Schoemaker, H.J.P., and Schimmel, P.R. (1977) Biochemistry 16, 5454-5460) attempts were made to find simple model systems in which this reaction might be studied in more detail. In the course of these investigations, it was found that Escherichia coli Ile-tRNA synthetase catalyzes cleavage of the glycosidic bond of 5-bromouridine. At pH 7.5, ambient temperatures, the turnover number is roughly 5/h. 5-Fluoro-, 5-chloro-, and 5-iodouridine are also cleaved in an analogous way by Ile-tRNA synthetase. In the case of uridine, conversion of uridine to uracil and ribose was also detected, but with a smaller turnover number. Three other E. coli and one mammalian aminoacyl-tRNA synthetases were also examined and all were found to catalyze glycosidic bond cleavage of 5-bromouridine. The data indicate that, in general, synthetases have a catalytic center that shows an unusual reactivity for uridine.

Mechanisms of killing of newborn larvae of Trichinella spiralis by neutrophils and eosinophils. Killing by generators of hydrogen peroxide in vitro.

Eosinophil and/or neutrophil leukocytes appear to have important roles in host defense against invasive, migratory helminth infestations, but the mechanisms of larval killing by leukocytes are uncertain. This study examines killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation with granule preparations of human eosinophils or neutrophils and generators of hydrogen peroxide (glucose-glucose oxidase) (G-GO) or superoxide and hydrogen peroxide (xanthine-xanthine oxidase). Larvae were killed by either hydrogen peroxide-generating system in a concentration-dependent manner. Direct enumeration of surviving larvae after incubation in microtiter wells containing the appropriate reagents was used in assess larval killing. Verification of the microplate assay was demonstrated by complete loss of larval ability to incorporate [(3)H]deoxyglucose and loss of infectivity after incubation in comparable concentrations of G-GO. Larvae were highly sensitive to oxidative products; significant killing occurred after incubation with 0.12 mU glucose oxidase and complete killing occurred with 0.5 mU. Comparable killing of bacteria required over 60 mU glucose oxidase. At 5 mU glucose oxidase, killing was complete after 6 h of incubation. Killing by G-GO was inhibited by catalase but not by boiled catalase or superoxide dismutase and was enhanced by azide. Addition of peroxidase in granule pellet preparations of eosinophils or neutrophils did not enhance killing by G-GO. These data indicate a remarkable susceptibility of newborn larvae of T. spiralis to the hydrogen peroxide generated by neutrophil and eosinophil leukocytes.

Effect of vitamin B6 deficiency on the levels of several water-soluble vitamins in tissues of germ-free and conventional rats.

The influence of vitamin B6 deficiency on the levels of several water-soluble vitamins and on acetyl-coenzyme A carboxylase activity was investigated using of germ-free and conventional rats. Judging from the vitamin B6 levels in tissues and the percent of decrease, the degree of vitamin B6 deficiency was more severe in the tissues of deficient germ-free rats than in deficient conventional rats. Nicotine acid, pantothenic acid and biotin levels per wet weight significantly decreased in the liver of vitaminB6-deficient germ-free rats, and nicotine acid levels per wet weight significant decreased in the liver of deficient conventional rats. In the kidney of vitamin B6-deficient germ-free rats, a significant decrease in riboflavin and biotin levels was observed, although there was no observable difference in riboflavin, nicotinic acid, biotin and patothenic acid levels in the kidney of deficient conventional rats. From an enzymatic standpoint, acetyl-coenzyme A carboxylase activity was especially significantly decreased in both germ-free and conventional rats fed a vitamin B6-deficient diet, and the percent od decrease was more in germ-free rats than in conventional ones. These findings suggest that vitamin B6 deficiency had stronger effects on the levels of water-soluble vitamins in germ-free rats compared with conventional rats.

Inter-relationships between solubilities, distribution coefficients and melting points of some substituted benzoic and phenylacetic acids.

Ten 4-hydroxy and 4-alkoxy benzoic and phenylalkanoic acids have been investigated. Solubilities in aqueous buffer at pH 1.2 were determined, together with distribution coefficients between the buffer and either octanol or isopropyl myristate. When plotted against the total number of carbon atoms in the side chains, log octanol/water distribution coefficients gave two parallel straight lines, one for the substituted benzoic acids, and the other for the substituted phenylalkanoic acids. The slopes approximated to 0.5, the generally accepted value for methylene. Similar plots could be obtained with isopropyl myristate, provided the hydroxy acid results were ignored, and also when log aqueous solubilities were plotted against carbon number, although there was considerable scatter. The differences between the distribution coefficient results were explained in terms of solute-solvent interactions, and the scatter attributed to variations in the heats of fusion of the solutes. Yalkowsky's equation (1977), linking aqueous solubilities and melting points with distribution coefficients, was applied to the results, and found to be of limited predictive value.

Calcium carrying system in the giant muscle fibre of the barnacle species, Balanus nubilus.

1. Single barnacle muscle fibres from Balanus nubilus were internally perfused with an isotonic solution containing 180 mM-tetraethylammonium acetate and the effects of Ca concentration in the external solution on the voltage-clamp currents, especially the initial inward current, were examined.2. Muscle fibre in external solution containing no added Ca (concentration of Ca < 10(-5)M) gave a current-voltage curve that showed a small inward current followed by a small outward current. An identical curve was obtained when the chloride in the external solution was replaced by acetate.3. The peak inward current increased with increase in Ca concentration in the external solution; but the increase in peak current for equal increments of Ca concentration was reduced and attained saturation conforming to an adsorption regime which can be described by the Langmuir equation.4. A double reciprocal plot of peak inward current due to Ca as a function of Ca concentration gave values of 20.7 mM and 200% (the current due to 20 mM-Ca normalized to 100%) for the dissociation constant and the maximum current respectively. These values were found to depend on the concentration of Mg used in the external solutions.5. The peak inward Ca currents measured at two Ca concentrations as a function of pH were unaffected in the pH range 6.5-8.8; however when the pH was reduced below 6.5, the currents were depressed.

[Characteristics of the changes in the intra- and extracellular K+ and Na+ concentrations and the intra- and extracellular pH in the area of cardiac ishemia in experimental myocardial infarct complicated by ventricular fibrillation].

Localized ischemia of the heart complicated by ventricular fibrillation is characterized by a tendency to a higher rate of decrease in intra- and extracellular K+ gradient and intracellular pH in the myocardium as compared to cases without fibrillation. The higher rate of K+ escape from the ischemic cells may be linked with a sharper intracellular oxidation, evidence of which is the correlative dependence between the severity of disorders of K+ balance and decrease in intracellular pH in the myocardium in ischemia.

[Proteolytic enzymes produced by Aspergillus ochraceus in relation to their plasma coagulating and fibrinolytic activities].

The paper describes some properties of coagulases and fibrinolytic enzymes isolated from a proteolytic complex produced by Aspergillus ochraceus HP-19 during submerged cultivation on a synthetic medium. Proteolytic enzymes with the plasmocoagulating activity hydrolyzed casein at the maximum rate at 45 degrees C and pH 8.0--10.0. The coagulases were stable at pH 5.0--7.0 and were rather resistant to low pH values. The enzymes were entirely inactivated at 55 degrees C within 20--30 min. The activity of the coagulases was inhibited with the ions of Cu, Co, Ag, Pb, Mn, Zn and Hg (1.10(-3) M) by 100, 91, 85, 50, 50, 38 and 25%, respectively. The coagulases were entirely inhibited with EDTA whereas PCMB and PMSF inhibited their activity only to a small extent. The mechanism for blood clotting with the coagulases of Aspergillus ochraceus HP-19 is presumed to consist in the activation of protrombin via its limited specific proteolysis. The fibrinolytic enzymes of Aspergillus ochraceus HP-19 had the optimal pH 8.5 for casein, were stable at pH 6.0, and entirely inactivated at 55 degrees C within 5 min. In contrast to coagulases, they were resistant to the action of heavy metal ions. The enzymes were stabilized by the ions of Ca. The activity of the fibrinolytic enzymes of Aspergillus ochraceus HP-19 was completely inhibited with PMSF. Therefore, they belong to the class of serine proteases.

Intracellular pH and the sodium requirement at fertilisation.

Several lines of evidence suggest that ionic messengers are primary agents in the metabolic derepression which occurs at fertilisation. The derepression at fertilisation or parthenogenetic activation of the sea urchin egg occurs in two main phases. The first phase, which triggers the early events of fertilisation, is mediated by transitory increase of intracellular free calcium. The second, which triggers the late events of fertilisation, is mediated by a rise in the intracellular pH (refs 4-6). The transition from the early events of fertilisation of sea urchin eggs to the late events requires a minimal concentration of sodium in the external medium. External Na+ is required for the acid effux which follows fertilisation. Na+ requirement and the acid effux have been correlated in a hypothesis which proposes that internal protons are exchanged for external Na+ (refs 8, 9). By using pH-sensitive microelectrodes, we have examined the relationship between external Na+ and internal pH more closely. We demonstrate here that the increase of the intracellular pH following egg activation does require external Na+. However, the relative insensitivity of the alkalisation of the egg cytoplasm to large reductions of external Na+ is evidence against the Na-H exchange hypothesis.

Controlled intravenous bicarbonate and fetal-maternal acid-base balance. I. The primipara.

The effects of a controlled sodium bicarbonate (SB) infusion on the acid-base balance of the primiparous mother and fetus at labor and delivery were evaluated. Two identical groups of primiparas with normal labor and delivery were studied. According to acid-base parameters observed in the mothers and fetuses of a control group, the pharmacologic dynamics, and the space of distribution of SB, 2 mEq/1 kg of total body weight were administered to the mothers of the study group, beginning at a cervical dilation of 6 cm until full dilation occurred. Highly significant changes in pH, base excess (BE), and plasma bicarbonate were observed in both the mothers and fetuses. In the latter, the significant changes appeared after a time lag of about 2 hours. No adverse effects in the mothers and fetuses were observed. The significant reduction of the relative fetal acidosis by the controlled SB infusion justifies further studies on the therapy potentials of this method in high-risk deliveries and during intrapartum fetal distress.

Ontogeny of human T cells.

Current knowledge of human T cell ontogeny is reviewed in terms of appearance of T cells in central and peripheral lymphoid organs, maturation of T cell markers, and development of immune functions. Extrapolation of growth curves derived from cell counts from fetal thymus, spleen, and bone marrow indicates the appearance of lymphocytes at 3.5 weeks gestation. E-rosette-forming cells are present in thymus at 11 weeks and in peripheral organs 15 to 16 weeks gestation. beta-2-Microglobulin is associated with all lymphoid cells by 13 weeks gestation. Lymphocyte responses to the T cell mitogen phytohemagglutinin (PHA) are first detected in thymus at 10 weeks and in spleen and blood 3 to 4 weeks later. Allogeneic responses in mixed lymphocyte reactions develop at about 7.5 weeks in fetal liver and later in thymus and peripheral organs. Lymphocytotoxicity for xenogeneic cells is a property of bone marrow cells and not thymocytes. Several aspects of development of a suppressor T cell in human neonates is discussed and related to similar findings in the mouse. These studies indicate a relatively high degree of maturation of human T cells during fetal life.

Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria.

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity in liver mitochondria decreases within 5 weeks to 0-6% of that of control animals fed on a diet supplemented with 0.5 ppm of selenium as sodium selenite. Analysis of the temperature dependence of energy-linked Ca(2+) uptake by means of Arrhenius plots reveals two breaks (at around 11 degrees C and 24 degrees C) in mitochondria isolated from selenium-supplemented animals, whereas in selenium-deficient rats the break at 11 degrees C is absent. Ca(2+)-loaded mitochondria of selenium-supplemented rats-i.e., with active glutathione peroxidase in the matrix-lose Ca(2+) rapidly, with a concomitant oxidation of endogenous NAD(P)H, when exposed to t-butyl hydroperoxide or H(2)O(2). In contrast, in selenium deficiency, t-butyl hydroperoxide and H(2)O(2) induce neither a release of Ca(2+) nor an oxidation of NAD(P)H. The peroxide-induced oxidation of NAD(P)H is reversible in the presence of succinate when no Ca(2+) has been taken up. When Ca(2+) has previously been accumulated, however, the oxidation of NAD(P)H is irreversible. Enzymatic analysis of mitochondrial pyridine nucleotides reveals that the peroxide-induced oxidation of NAD(P)H in Ca(2+)-loaded mitochondria leads to a loss of NAD(+) and NADP(+). It is proposed that the redox state of mitochondrial pyridine nucleotides can be or is in part controlled by glutathione peroxidase and glutathione reductase and is a factor in the balance of Ca(2+) between mitochondria and medium.

Estimates of quantal content during 'chemical potentiation' of transmitter release.

The number of quantal transmitter packets (m), released from motor nerve terminals in response to a single stimulus, has been estimated from the ratio of the amplitudes of endplate currents (e.p.c.) to spontaneous miniature endplate currents (m.e.p.c.), in voltage-clamped endplates of the frog. At 6 degrees C, the average value of m at normal nerve-muscle junctions was about 300. If allowance is made for the temporal dispersion of quantal transmitter release during the e.p.c., this value is increased by about 30%. After treatment with diaminopyridine or tetraethylammonium, transmitter release in response to a nerve stimulus is greatly enhanced and values of m exceeding 10(4) are frequently found. Moreover, the duration of the e.p.c. becomes much longer than that of the m.e.p.cs. The number of packets then liberated during the e.p.c. is much larger than the number of 'active zones' of the endplate and may even exceed the total number of vesicles lined up in twin-files adjacent to the presynaptic membrane.

Effects of MIF-I and melatonin on novelty-induced defecation and associated plasma 11-OHCS and brain catecholamines.

In two experiments the effects were investigated of MSH-inhibiting factor-I (MIF-I) and of Melatonin on step-down latencies, defection, plasma 11-OHCS levels, whole brain DA and whole brain NE concentrations on Days 1, 3 and 5 of novelty exposure. Treatment with MIF-I led to a significant habituation of novelty-induced defecation over 5 days, whereas plasma 11-OHCS level was reduced only on Day 1. The concentrations of whole brain DA and whole brain NE also showed a significant increase over days of MIF-I and novelty treatment. Melatonin treatment, on the other hand, significantly inhibited novelty-induced defecation and reduced plasma 11-OHCS level on Day 5 of novelty exposure. Melatonin treatment led to a significant increase of whole brain DA in animals exposed to novelty for 5 days. Neither MIF-I nor Melatonin was found to significantly affect the step-down activity of treated animals. The overall results suggested a possible relationship between novelty-induced defecation and brain DA levels of MIF-I and Melatonin treated animals.

Discriminative stimulus properties of pentylenetetrazol and bemegride: some generalization and antagonism tests.

In an operant procedure of lever pressing on a FR 10 schedule of food reinforcement, male hooded rats were trained to respond with a lever on one side of a food cup following a drug injection, and to respond with a lever on the alternate side following a 1 ml/kg saline injection. All of 14 subjects learned to discriminate reliably between the effects of 20 mg/kg pentylenetetrazol (PTZ) and saline. Seven of eight rats learned to discriminate between the effects of bemegride (5 mg/kg) and saline. None of 14 rats learned to discriminate between 5mg/kg PTZ and saline. The bemegride discriminative stimulus generalized to PTZ (20mg/kg) and was antagonized by chlordiazepoxide (10 mg/kg). Chlordiazepoxide, diazepam, flurazepam, clobazam, and meprobamate were all effective antagonist of PTZ in a dose-dependent manner. Bemegride and cocaine generalized to the PTZ discriminative stimulus in a dose-dependent manner, but d-amphetamine, methylphenidate, and nicotine did not. Since bemegride and PTZ are convulsants at higher doses, the discriminative stimulus properties of these drugs might be based on a subtle convulsive brain state. The anxiolytic properties of benzodiazepines and meprobamate suggest that the discriminative stimulus produced by these convulsants is related to an "anxiety-inducing" action.

Temperature and acid-base status of human blood at constant and variable total CO2 content.

The influence of temperature on the acid-base status of normal human blood was studied in closed systems (constant CCO2) and open systems (variable CCO2). pH-T coefficients of true plasma and erythrocytes in closed systems were similar to coefficients for water (dpH/dT = -0.017 U/degrees C at 25 degrees C). Between 26 degrees C and 42 degrees C there was no significant variation in the relative alkalinities [OH-]/[H+], the charge state of proteins in plasma and erythrocytes or the proton Donnan ration. The equations established enabled calculation of the pH of true plasma and erythrocytes and of blood PCO2 and temperature, using only one of these four parameters. Under open-system conditions, temperature was shown to cause a rise in the apparent buffer power of whole blood non-bicarbonate systems (28.8 and 34.8 mM.l-1.u-1 at 26 degrees C and 42 degrees C respectively). These results show (1) that erythrocytes in closed systems seem very well able to maintain proton distribution regardless of temperature fluctuations and (2) when blood temperature rises, it cannot be excluded that vital organs are better protected against respiratory disturbances.

Modification of cerebrovascular CO2 reactivity by inhibition of dopamine beta-hydroxylase.

The influence of sympathetic nervous activity on cerebral circulation and cerebrovascular CO2 reactivity was investigated through inhibition of dopamine beta-hydroxylase (DBH). A PO2 electrode, a PCO2 electrode and a plate-type thermocouple-flowmeter were placed on the pial surface of the cat brain. Cerebrocortical PO2, PCO2, cerebrocortical blood flow and arterial blood pressure were continuously recorded before, during and after intracarotid infusion of 10 mg/kg of fusaric acid, a potent DBH inhibitor. The effects of 5% CO2 inhalation and hyperventilation were measured before and after the inhibition of DBH. Following the intracarotid infusion of fusaric acid, cerebrocortical PO2 and cerebrocortical blood flow increased significantly. After the inhibition of DBH, the degree of the increase in cerebrocortical PO2 during 5% CO2 inhalation was enhanced while the degree of the decrease in cerebrocortical PO2 during hyperventilation did not show any significant change. The cerebral vasodilatation caused by fusaric acid suggests that the sympathetic nervous system takes part in the resting tone of cerebral blood vessels. The increase in the cerebrovascular CO2 reactivity produced by the inhibition of DBH suggests that the sympathetic nervous system modifies cerebrovascular CO2 reactivity.

[Heterogeneity and regulation of glutamate dehydrogenase activity in mammalian brain and liver].

The present report concerns the study of the catalytic properties and the coenzyme affinity of glutamate dehydrogenase (GDH) and its isoenzymes in various preparations of the brain and liver as well as the different regulatory mechanisms controlling the ratio of the rates of biogenesis and breakdown of glutamate (Glu). The investigations carried out showed that GDH activity of various preparations of brain and liver (crystalline enzymes, cellular extracts and mitochondria) are markedly different from each other by their catalytic and regulatory properties as well as by their coenzyme activity. The data obtained make us conclude that nicotinamide-hypoxanthine-nucleotide (deaminoNAD) is a more effective coenzyme in the oxidative deamination of Glu, than other piridine nucleotides (NAD, NADP, deamino-NADP). It is supposed that in the formation of ammonia and amino acids in brain and especially liver, together with other known mechanisms an important role may be ascribed to the process of transdeamination. In this aspect, as a co-factor of oxidative deamination of Glu deamino-NAD (D-NAD) is thought to be of significant importance.

Renal proximal tubular acidification. Role of brush-border and cytoplasmic carbonic anhydrase.

Carbonic anhydrase is found in the cytoplasm and brush border membranes of renal proximal tubular cells. Both the soluble and the membrane-bound enzyme have been assigned roles for the secretion of hydrogen ions into the tubular fluid and hence also for the reabsorption of bicarbonate. Attempts were made to differentiate between the roles of these enzymes for the rate of proximal tubular acidification. Proximal tubules of rats were instilled and perfused with bicarbonate solutions containing carbonic anhydrase inhibitors, especially designed to be impermeable to cell membranes. The acidification rate was measured with an antimony micro-electrode system--the only instantly responding micro-pH electrode. The membrane impermeable inhibitors had no effect on this rate in contrast to acetazolamide, which markedly inhibited the acidification rate when administered intraluminally. It is therefore concluded that the cytoplasmic carbonic anhydrase is the important enzyme for the proximal tubular acidification rate, and hence the rate of bicarbonate reabsorption. The function of the brush border enzyme remains an outstanding problem.

Lithium effects: relation to lithium dose and to plasma peak levels.

In a 24-hour study, plasma peak lithium was determined in manic-melancholic patients who routinely had their entire lithium dose at night. A correlation analysis was undertaken of the relation of plasma peak level and the dose of lithium to a number of lithium induced changes: Increase in urine volume, weight gain, decrease in plasm phosphate, increase in plasma magnesium, decrease in plasma urea, increase in plasma alkaline phosphatase, increase in urinary pH. Only the changes in plasma phosphate and in urine pH were significantly correlated to the peak value of plasma lithium. The increase in urine volume was significantly correlated to the dose of lithium.

Blood and ruminal fluid profiles in carbohydrate-foundered cattle.

The relationships of acetylhistamine and histamine to the clinical signs of carbohydrate-induced acidosis were investigated in beef steers. Blood pH and plasma L-lactic acid decreased and serum sodium, serum potassium, ruminal fluid L-lactic acid, ruminal fluid histamine, and ruminal fluid and blood acetylhistamine increased in carbohydrate-engorged steers as compared with the changes in the steers while feeding on pasture (forage-fed steers). Twelve to 14 hours after the steers had become engorged, clinical signs of laminitis ("feedlot founder") were observed in three of six steers. These signs appeared 4 to 6 hours after blood acetylhistamine attained maximal concentration (2.9997 +/- 1.7054 microgram of histamine base/ml of blood) and blood pH decreased to 7.260 +/- 0.026 at 8 hours after engorgement. Blood histamine value reached 0.1298 +/- 0.1095 microgram of histamine base/ml 4 hours after engorgement (8 to 10 hours before the appearance of clinical illness), but had reached maximal concentration 32 hours after engorgement (0.3300 +/- 0.028 microgram of histamine base/ml of blood).

[Unstable angina with threatening coronary lesions turned down for surgery. Outcome and prognostic factors].

The aim of this study based on a series of 200 patients, was to define the outcome and the prognostic factors of patients presenting with unstable angina, according to Bertolazi's criteria [3] and at least one stenosis greater than 80% on a proximal segment of a main coronary trunc, and to determine which factors should eventually be taken into consideration in the discussion of surgical indications. 70 out of 200 patients (35%) were turned down for direct revascularisation surgery because of an ejection fraction less than 0,35 and/or a poor arterial run off. Coronary arteriography showed 30% patients with a menacing stenosis (greater than 80%) on all three vessels, 36% on two vessels and 22% on a single vessel. The distribution and the extent of the lesions was about the same as in the operated patients. 20% patients had an ejection fraction less than 0,35, 24% between 0,34 and 0,50, and 56% greater than 0,50. At patient, the follow up period ranges from 22 to 66 months (average 32 months). In this group, the hospital mortality was 2,9%, the secondary cardiac deaths 16% and the global mortality 19% compared to 12,6% for the operated patients in the same period. The incidence of secondary non-fatal infarction was low (9%). 52% of survivors have persistent angina, 39% severe (Class II or III). Two prognostic factors were detected from this study: the type of angina: the intermediary syndrome had a bad prognosis, 38,5% mortality compared to 13% for aggravated chronic angina; and the ventriculography: patients with ejection fractions less than 0,35 had 64% mortality compared to 7,3% for those with ejection fractions greater than 0,40. The number of menacing lesions, the extent of the lesions of the artery involved did not affect the prognosis when severe abnormalities of left ventricular function were absent.

Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins.

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie--Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.

The effects of alpha- and beta-adrenergic receptor blockers on the pressure responses to isometric exercise in hypertensive patients.

1. The cardiovascular responses to handgrip exercise have been studied in ten patients with uncomplicated essential hypertension in a randomized crossover study of propranolol and prazosin. 2. Isometric handgrip exercise was performed with a calibrated strain gauge dynamometer at 30% of maximum voluntary contraction for 3 min. 3. Blood pressure and heart rate were measured in the supine position at rest and in the last 10 s of the exercise period. 4. These exercise studies were undertaken at the end of a run-in period and at the end of 1 month's optimal therapy with the two drugs. 5. The active treatment periods were separated by a 2 weeks placebo washout period. 6. Both drugs lowered the supine and standing systolic and diastolic pressures and there was no difference between these drugs in their effect on these variables. 7. Propranolol lowered the resting heart rate and neither drug suppressed the pressor response to isometric exercise. 8. The degree of pressure rise was similar with both drugs but propranolol suppressed isometric exercise-induced tachycardia.

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum.

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360-410mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores. The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C340) which has an oxidation-reduction midpoint potential close to 340 mV (n=1) at pH 6.0--7.6. The Em of this component varies by 60 mV/pH unit between pH 7.6 and 9.2. The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C340.

Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.

Characterization of rat testicular guanylate cyclase during development.

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.

The resolution of dopamine and beta 1- and beta 2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex.

The stimulation of adenylate cyclase by dopamine and various beta-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the beta-arenergic receptors coupled to adenylate cyclase could be classified as either beta 1 and beta 2 subtypes in the different regions studied. The stimulation of adenylate cyclase by the beta-adrenergic agonist, (-)isoproterenol (5 X 10(-6) M), was completely blocked by the specific beta-adrenergic antagonist, (p)alprenolol (1-(-5) M), but not by the dopaminergic antagonist, fluphenazine (10(-5) M), whereas the stimulation of adenylate cyclase by (-)epinephrine (10(-4) M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (-)epinephrine effect was always blocked in the combined presence of (p)alprenolol and fluphenazine. The adenylate cyclase stimulation by (p)epinephrine which is not blocked by (p)alprenolol was due to interaction of (p)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum. Regional differences in the affinity of beta-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10(-5) M). In the cerebellum the potency order was (+/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (+/-)isoproterenol greater than (-)epinephrine greater than (+/-)salbutamol greater than (-)norepinephrine, indicating the presence of a beta 2-adrenergic receptor. In the cerebral cortex the potency order was (-)isoproterenol greater than +/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (-)epinephrine = (-)norepinephrine ((+/-)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a beta 1-adrenergic receptor in these two regions. (+/-)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex. The ratio of the antagonist potencies of (+/-)practolol and (+/-)butoxamine preferential beta 1- and beta 2-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a beta 2 subtype of receptor coupled to adenylate cyclase in the former and beta 1 subtypes in the latter two regions. The comparison between the affinities of a series of beta-adrenergic agonists and antagonists for the beta-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized beta 2-adrenergic receptors in lung and beta 1-adrenergic receptor in heart substantiated this conclusion.

Human leucocyte cerebroside sulphate sulphatase.

Some of the properties of the cerebroside sulphate sulphatase of human leucocyte extracts have been studied. The enzyme has an apparent KM of 2.9 mmol/l and is inhibited by the products of the reaction, sulphate and galactocerebroside. A number of divalent metal ions including manganese and magnesium stimulated the reaction only slightly at 5 mmol/l but inhibited strongly at 20 mmol/l. Triton X-100 present in leucocyte extracts also inhibited, increasing both the apparent KM and Vmax. The enzyme activity was dependent on the presence of anionic detergents. At low substrate concentrations a crude taurocholate preparation was the most active of all bile acids examined. Pure taurocholate and sodium cholate were considerably less active. At highter substrate concentrations however, sodium cholate produced the greatest stimulation of enzyme activity. These data suggest that the bile acid/substrate ratio may be a critical factor in determining cerebroside sulphate sulphatase activity at different substrate concentrations.

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.

Screening in diabetes mellitus: report of the Atlanta workshop.

A Diabetes Screening Workshop was held in Atlanta, Georgia, May 15--17, 1978, which was sponsored by the Center for Disease Control, the American Diabetes Association, and the National Institute of Arthritis, Metabolism, and Digestive Diseases. The workshop formulated the following recommendations for the use of screening procedures in diabetes mellitus from a community control viewpoint: (1) screening for asymptomatic glucose intolerance should be done among pregnant women as part of a well-coordinated program to decrease perinatal morbidity and mortality; (2) screening programs to detect asymptomatic glucose intolerance per se are not recommended as health services in nonpregnant populations; (3) screening for diabetes or its complications for research purposes should be done only as part of well-designed studies focusing on identification of predictive factors, implementation and effectiveness of preventive and therapeutic measures, descriptive epidemiology in selected populations, dynamic and economic factors of the medical care system related to case detection and management, and the nature and effects of screening processes; (4) information and education programs for health care providers, parents, and the general public should be implemented to bring about increased awareness of the clinical signs and symptoms of diabetes; and (5) all persons known to have diabetes should be evaluated regularly for the detection and management of the common chronic complications of the disease.

Bovine serum chitinase.

1. A glycol-chitin-splitting enzyme without lysozyme (muramidase) activity has been found in calf serum. The enzyme also degrades colloidal chitin and is thus a true chitinase, 1,4-beta-poly-N-acetylglucosaminidase, without exo-beta-N-acetylglucosaminidase effect. 2. The enzyme is purified 1000-fold by ion-exchange chromatography and gel filtration. Its optimal activity is between pH 1.5-2.0 with glycol chitin and between pH 3-6 in a rather broad optimum with colloidal chitin as substrate. The optimal stability of the enzyme is in the pH interval 3.0-6.5 when tested by incubation with glycol chitin at 50 degrees C for 60 min. The optimal temperature for the degradation of glycol chitin is 40 degrees C when assayed at pH 1.5 and 51 degrees C when assayed at pH 3.5. 3. The enzyme is activated by moderate heating at pH 6.5. The highest relative activity, 135% is reached after 45 min incubation at 30 degrees C, pH 5 or after 30 min at 40, pH 2.4. By incubation with small amounts of trypsin at pH 6.5 at 3m degrees C the enzyme was temporarily activated. 4. The isoelectric point, pH 5.3, and the molecular weight, 47,000 +/- 3,000 were determined by respectively isoelectric focusing and gel filtration. 5. The Michaelis-Menten constant, Km = 0.76 +/- 0.05 (S.E.) mg/ml, was measured with glycol chitin as substrate.

Antiulcer activity of hypertonic solutions in the rat: possible role of prostaglandins.

The effects of hypertonic solutions on gastric acidity and on experimental gastric and duodenal ulcers as well as on gastric prostaglandins (PGs) were studied in the rat. The oral administration of a 10% NaCl solution resulted in complete absence of free acidity and very significant reductions in total acidity 24 h after pyloric ligation. The antiulcer effect of hypertonic saline was studied in three experimental models. In pyloric-ligated rats, both the incidence and the severity of gastric ulcers were remarkably reduced by hypertonic saline treatment. Indomethacin-induced gastric erosions were significantly reduced by hypertonic NaCl or sorbitol and completely prevented by hypertonic xylitol. Cysteamine-induced duodenal ulcers were also significantly reduced by hypertonic solutions of NaCl, xylitol or sorbitol. In the latter model, indomethacin potentiated the ulcerogenic effect of cysteamine and also reduced the efficacy of the hypertonic NaCl gavage. The possible contribution of PGs to these effects was further investigated by analysing PGE in the gastric mucosa and juice. Rats treated orally with hypertonic NaCl solutions had several-fold higher PGE contents in their gastric mucosa as well as higher PGE levels in the gastric juice. It is concluded that hypertonic solutions stimulate endogenous PGE biosynthesis and also exert profound antiulcer effects in the rat. A causal relationship between the two phenomena is suggested.

The midwife and the family unit.

The activities of the midwife dealing with home deliveries or working in a ward and her relationship with pregnant women are discussed.

Reduction of cinerulose in aclacinomycin-A by soluble and microsomal cinerulose reductases.

The in vitro metabolism of the antitumor anthracycline antibiotic, aclacinomycin-A, was studied using rat liver homogenate. In the presence of NADH or NADPH, aclacinomycin-A was converted to aclacinomycin-A analogs, MA144 M1 and MA144 N1, which were stereospecifically reduced at the keto group of the C-4''' position of L-cinerulose in aclacinomycin-A. Subcellular fractionation indicated that the production of MA144 M1, which was reduced to L-amicetose, was catalyzed by NADPH-dependent soluble cinerulose reductase I, and the production of MA144 N1, which was reduced to L-rhodinose, was catalyzed by NADPH-dependent soluble cinerulose reductase II and NADH-dependent microsomal cinerulose reductase. The properties of these three enzymes were studied. Soluble cinerulose reductase I which produces MA144 M1 showed a optimum pH at 6.3, Km values of 3.3 x 10(-4) M for aclacinomycin-A and 3.2 x 10(-5) M for NADPH. Soluble cinerulose reductase II which produces MA144 N1 showed a pH optimum at 6.3 and Km values of 2.0 x 10(-3) M for aclacinomycin-A and 4.0 x 10(-5) M for NADPH. All thesse reductases were sensitive to sulfhydryl reagents and were inhibited by vitamin K3. Microsomal cinerulose reductase showed sensitivity to diconmarol and ferrous ion. The main nondegradative pathways of aclacinomycin-A were discussed from these results.

Lithium-induced nephrogenic diabetes insipidus: studies of tubular function and pathogenesis.

We describe a patient with lithium-induced nephrogenic diabetes insipidus in whom detailed investigations of distal tubular function were performed. Clearance of free water during water diuresis was found to be augmented. This suggests proximal suppression of sodium reabsorption by lithium. Reabsorption of free water during high solute clearance was impaired. Acidification of the urine following ammonium chloride loading was abnormal, and this was corrected by sodium sulfate infusion. The cellular mechanism of lithium was investigated by means of indomethacin, an inhibitor of prostaglandin synthesis. Indomethacin caused a partial reversal of the nephrogenic diabetes insipidus, suggesting that the primary cellular action of lithium may be to inhibit the formation of cyclic AMP in the collecting duct cell, although a direct action of indomethacin in increasing solutes in the renal medulla could not be ruled out. It is possible that the lithium-induced polyuria is partially due to an enhancement by lithium of renal prostaglandin action.

Aortic body chemoreceptor responses to changes in PCO2 and PO2 in the cat.

Responses of aortic chemoreceptor afferents to a range of arterial carbon dioxide tension (Paco2) changes at various levels of arterial oxygen tension (Pao2) were investigated in 18 cats anesthetized with alpha-chloralose and maintained at 38 degrees C. Aortic chemoreceptor activity, end-tidal oxygen pressure, end-tidal carbon dioxide pressure, and arterial blood pressure were continuously monitored. Arterial blood gases were measured in steady states. Single or a few clearly identifiable afferents were studied during changes and steady states of Pao2 and Paco2. All the aortic chemoreceptor afferent discharge rates increased with Paco2 increases from hypercapnia (10-15 Torr) to normocapnia and moderate hypercapnia (30-50 Torr) and with Pao2 decreases from above 400 to 30 Torr. Hypoxia augmented the response to Paco2 most effectively in the range of 10-40 Torr. At any Pao2, the discharge rate reached a plateau with sufficient intensity of hypercapnia. The Paco2 stimulus threshold at a Pao2 of 440 Torr was about 15 Torr, and at a Pao2 of 60 Torr it was 10 Torr. In the transition from hypocapnia to hypercapnia, responses increased gradually, usually without an overshoot. The steady-state responses to Paco2 of the majority of aortic chemoreceptors resembled those of carotid chemoreceptors. The responses of both receptors can be attributed to the same basic type of mechanism.

Purification and characterization of an endonuclease specific for single-stranded DNA from Bacillus subtilis Marburg.

Bacillus subtilis Marburg TI (thy,trpC2) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease MII), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000:1 by comparing the initial rates of introducing single-strand breaks into the DNA's.

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae.

Diaphragmed fenestrae (DF) are sites of increased vascular permeability. The anionic charge distribution at the luminal aspect of the DF of the endothelium of the bone marrow vessels has been studied after aldehyde fixation by means of colloidal iron (CI), native ferritin (NF), and polycationic ferritin (PCF). At pH 1.8, these cationic agents are bound by the nonmodified luminal endothelial cell surface but not at the sites of the DF. PCF was used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels, whereas NF which has a pI of 4.5 is anionic above this point). PCF shows increased binding at the DF from pH 3.5 upwards. PCF binding at pH 1.8 at the nonmodified luminal cell surface is significantly diminished by neuraminidase treatment which, however, does not perceptibly reduce PCF binding at the higher pH levels. It is concluded that there are exposed sialic acid groups at the lunimal cell surface which are absent or significantly fewer at the sites of the DF, whereas other anionic materials possibly with a pKa higher than that of sialic acid (pKa 2.6) are present both at the DF and at the nonmodified endothelial cell surface.

Long-term use of a "stable" bicarbonate containing dialysate.

We present an original method for the preparation of "stable" dialysate containing 35 mEq/l of bicarbonate. The dialysate was utilized with 4 patients for periods ranging from 4 months to 1 year according to a short-term recirculated dialysis schedule in closed circuit (20-40L) (2-2 1/2 hrs) on alternate days. Preliminary results are reported here with respect to the tollerance of the dialytic run and correction of the acid-base balance equilibrium. The clinical tollerance is excellent despite high dehydration rates even in patients particularly sensitive to ultrafiltration. The acidosis correction would seem to be much better with bicarbonate than with traditional dialysis. The difference is even higher if we consider the brevity of the dialysis. During the bicarbonate dialysis we do not observe any fall of the PCO2 or significant difference in PO2 in the patient's blood. The correction of acidosis probably causes the normalization of pre-dialytic potassiemia in spite the "net" removal of K with short dialysis is considerably less.

Membrane potential oscillations in molluscan "burster" neurones.

Membrane potential oscillations can be induced in molluscan neurones under a variety of artificial conditions. In the so-called 'burster' neurones oscillations are generated even in isolated cells. A likely mechanism for 'bursting' involves the following ionic currents: 1. A transient inward current carried by Na+ and Ca2+. This current is responsible for the upstroke of the action potentials. 2. A delayed outward current carried by K+. This current is voltage-sensitive and is responsible for the downstroke of the action potential during the early part of the burst. It becomes progressively inactivated during the burst. Its amplitude depends on the intracellular pH. 3. A rapidly developing outward current carried by K+ which is inactivated at potentials close to action potential threshold. This current tends to hold the membrane in the hyperpolarized state and is involved in spacing the action potentials. 4. A prolonged inward current which may not inactivate. It is probably carried by both Na+ and Ca2+. This current is responsible for the depolarizing phase of the burst but also contributes to the action potential. 5. A slowly developing outward current, carried by K+. This current appears as a result of a slow increase in intracellular ionized calcium and is responsible for the hyperpolarizing phase of the burst. Note that a transient increase in this current may also contribute to the falling phase of the action potential during the later stages of the burst. It is also sensitive to intracellular pH. One of the more significant features of this system of producing membrane potential oscillations is that the frequency of the bursts depends on the rate at which the intracellular ionized calcium returns to its resting level. This process depends on the metabolic state of the animal which can thereby exert a considerable influence on the electrical activity of burster neurones.