Bromocriptine in hyperprolactinemic amenorrhea: a possible early effect on ovarian steroidogenesis. The effect of bromocriptine was studied in a 28-year-old cretin woman who presented with long-term untreated primary hypothyroidism, primary amenorrhea, hyperprolactinemia, and an enlarged pituitary fossa. Bromocriptine therapy lowered the plasma prolactin and caused an early rise in plasma 17 beta-estradiol levels and uterine bleeding, despite the failure of plasma gonadotrophins to respond to luteinizing hormone release hormone (LHRH) administration. Thus, it is suggested that bromocriptine may have an early direct effect on the ovary in hyperprolactinemic amenorrhea.


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.


Cell mediated and local immunity to spermatozoa in infertility. The possibility of the immunological factor as an etiological cause of infertility was investigated in 50 infertile couples. Of these, 31 couples were considered as an "unexplained sterility" group, while in the other 19 couples a variety of disorders were diagnosed. In vitro, leucocyte migration inhibition test (LMIT) was used for the detection of female cell-mediated immunity against sperm antigens. Sperm immobilization test (SIT) and sperm toxicity tests (STT) were used for the detection of antisperm antibodies in the cervical mucus. Out of the 31 "unexplained sterility" cases, six revealed a positive LMIT (5.3%). In 26 couples with an abnormal postcoital test (PCT), 10 cases had a positive SIT and STT (38.5%). However, out of the group of 16 couples with a normal PCT only in one case was a positive SIT and STT found (6.2%). The potential role of immunological reactions in infertility will be discussed.


Reaction of azide radicals with amino acids and proteins. The azide radical N3 reacts selectively with amino acids, in neutral solution preferentially with tryptophan (k (N3 + TrpH) = 4.1 X 10(9) dm3 mol(-1s-1) and in alkaline solution also with cysteine and tyrosine (k(N3 + CyS-) = 2.7 X 10(9) dm3 mol-1s-1) and k(N3 + TyrO-) equals 03.6 X 10(9) dm3 mol-1s-1). Oxidation of the enzyme yADH by N3 involves primary attacks, mainly at tryptophan residues, and subsequent slow secondary reactions. N3-induced inactivation of yADH is likely to occur upon oxidation of tryptophan residues in the substrate binding pocket (58-TrpH and 93-TrpH) since the substrate ethanol although unreactive with N3, protects yADH and since elADH, which does not contain tryptophan in the substrate pocket, is comparatively resistant against N3-attack. N3 exhibits low reactivity with nucleic acid derivatives and it is inert towards aliphatic compounds such as methanol and sodium dodecylsulphate.


Calcium oxalate dihydrate crystal growth. Calcium oxalate dihydrate (COD), although thermodynamically unstable with respect to calcium oxalate monohydrate (COM), is more commonly seen in human urine. For the first time, a COD-seeded crystal growth assay has been developed. Seed crystals of COD were precipitated from filtered urine by the addition of ammonium oxalate and were stored dry and added to a supersaturated calcium oxalate solution to initiate an experiment. The growth rate in the COD-seeded system was 22 per cent of that for a COM-seeded system, for equivalent surface areas. Urine samples from normal subjects have similar inhibitory activity in the COD- and COM-seeded systems, as do pyrophosphate and heparin, which are known inhibitors of COM crystal growth. These results indicate that the technically simpler COM-seeded growth assay is suitable for measurement of inhibitors of calcium oxalate growth in urine.


Metabolic heterogeneity of muscle fibers classified by myosin ATPase. Muscle fibers are commonly classified histochemically into three types by the staining intensity for myosin ATPase combined with those for metabolic enzymes. Preincubation at pH 4.6 gives rise to three staining intensities of myosin ATPase which are also used for fiber typing. The two classification systems were compared by computer analysis of the individual staining profiles of over 2,500 fibers, and found not to be equivalent. The analysis showed metabolic heterogeneity among the fiber groups distinguished according to their differences in myosin ATPase.


Histochemical study on the pathogenesis of chlorocyclizine-induced pulmonary lipidosis. Male rats were fed a diet containing chlorocyclizine in high concentrations for about 3 weeks. They lost weight and showed respiratory distress. The lungs contained clusters of foam cells in the alveoli. Acid esterase staining revealed reduction of activity in alveolar cells presumed to be granular pneumocytes and absence of activity in the foam cells. The lipid showed in the foam cells could not be stained with Sudan dyes, except at high temperature, and was not stained by phospholipid and cholesterol procedures. This indicated that the stored lipids are probably solid at room temperature, consisting of saturated triglycerides and/or phospholipids. It is suggested that the lipid originated in the granular pneumocytes. The drug might have deranged the esterase-phospholipase activity in these cells and in the macrophages.


Community health nursing in an urban day care center. This paper has endeavored to depict some of the student achievements during community health nursing practice in a less traditional clinical setting, a day care center for infants and toddlers. There appear to have been a number of advantages to such a clinical placement. For one thing, students had the opportunity to devise a package of preventive/maintenance health services for a specific group of healthy children. This they were able to implement in collaboration with the day care center staff and the children's families. In the course of delivering the health services, they learned firsthand the strategies inherent in planning and negotiating among one another. In recognition of the integrity of child health as a component of family health care, they accounted regularly to the parents in both individualized and group fashion. Through the experiences over the course of the quarter, there seemed to be a heightened awareness of the potential for innovative nursing practice in urban communities.


Pharmacologic management of circulatory shock: cardiovascular drugs and corticosteroids. Effective use of cardiovascular drugs in the management of circulatory shock requires knowledge of the pathophysiologic changes occurring in the different types and stages of shock and an understanding of the specific hemodynamic actions of drugs used to correct shock. Objectives of therapy are to: (1) restore circulating blood volume, (2) ensure of increase perfusion of critical organs by selectively reducing arteriolar resistance, (3) augment cardiac output, and (4) increase perfusion pressure. If blood volume is not restored, drugs may be ineffective and can even induce deleterious effects. If reflex sympathetic discharge has induced generalized vasoconstriction, it is irrational to expect beneficial results from administration of exogenous vasconstrictor agents. Instead, selective vasodilatation of vascular beds in critical tissues (eg, myocardium, intestines, and kidneys) accompanied by increased perfusion pressure and cardiac output can prove useful. Large doses of adrenocorticosteroids are used commonly in the therapy of different shock conditions, based primary on empiric tests of efficacy. Although such steroids may not influence the course of shock by direct cardiovascular effects, beneficial actions may result from mechanisms such as protection of cell membrane integrity and stabilization of lysosomes.


Metabolic and electrolyte disturbances in acute canine babesiosis. Arterial blood pH, PCO2, bicarbonate, base excess/deficit, and lactate, as well as serum sodium, potassium, and chloride were measured in clinically normal dogs and in dogs with acute canine babesiosis. Metabolic acidosis developed in dogs with fatal as well as nonfatal Babesia canis infection. In the fatal group, the acidosis was uncompensated; among survivors, base deficit and blood lactate were significantly lower, and pH, PCO2, and bicarbonate values were significantly higher. Serum potassium values were significantly lower, and serum chloride values were significantly higher in dogs with acute babesiosis than in clinically normal dogs. The shock resulting from acute canine babesiosis is best viewed as anemic shock. Treatment should include an alkalizing agent, a blood transfusion, fluid therapy, and a babesicidal drug.


Purification and properties of a nicotinamide adenine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism. Glycerol:NAD+2-OXIDOREDUCTASE (EC 1.1.1.6) was purified to homogeneity from a mutant of Escherichia coli K12 that uses this enzyme, instead of ATP:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit of 39,000 daltons. During electrophoresis under nondenaturing conditions, the protein migrates as two bands. These two forms, both of which are enzymatically active, appear to be dimers and octomers of the same subunit. The optimal pH for the oxidation of glycerol is about 10, and that for the reduction of dihydroxyacetone is about 6. Glycerol dehydrogenation is highly activated by NH4+, K+, or Rb+, but strongly inhibited by N-ethylmalemide, 8-hydroxyquinoline, 1,10-phenanthroline, Cu2+, and Ca2+. The enzyme exhibits a broad substrate specificity. In addition to glycerol, it act on 1,2-propanediol and several of its analogs.


Membrane H+ conductance of Streptococcus lactis. Membrane conductance to H+ was measured in the anaerobic bacterium Streptoccus lactis by a pulse technique employing a low driving force (0.1 pH unit; 6 mV). Over the pH range of 3.7 to 8.5, a constant value for passive H+ conductance was observed, corresponding to 0.2 mumol of H+/s per p/ unit per g, dry weight (1.6 microS/cm2 of surface area). The pH insensitivity of this low basal H+ conductance supports the idea that a circulation of protons can mediate highly efficiency engery transductions across the membranes of bacteria.


Distribution of coenzyme F420 and properties of its hydrolytic fragments. The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.


Modifiers of heterocyst repression and spacing and formation of heterocysts without nitrogenase in the cyanobacterium Anabaena variabilis. Twelve amino acid analogs and related compounds were screened for their ability to induce heterocysts in ammonia-repressed, undifferential filaments of Anabaena variabilis. As has been previously described, 1-methionine-dl-sulfoximine induces both heterocysts and nitrogenase. In contrast, dl-7-azatryptophan and beta-2-thienyl-dl-alanine were found to induce heterocysts but not nitrogenase activity (measured as acetylene reduction) even under microaerobic conditions. When the initial ammonium concentration was reduced, dl-7-azatryptophan-treated cultures sequentially produced heterocysts and then nitrogenase activity, but nitrogenase was detected only when a parallel culture without analog also became capable of acetylene reduction. Neither of the two latter analogs affected gamma-glutamyl transferase activity in crude extracts. All three analogs significantly reduced the mean interheterocyst distance in nitrogen-fixing cultures.


Characterization of the Bacillus subtilis motile system driven by an artificially created proton motive force. Transient swimming was induced in energy-depleted cells of Bacillus subtilis by an artificial proton motive force, which was created by valinomycin addition and a pH reduction. This system did not require any ions except protons in the medium. The size of the induced motility was strongly influenced by changes in the size of either the K+ diffusion potential or the pH gradient. A rough estimation indicated that a proton motive force higher than -100 mV was required for induction of translational swimming of the cell. Corresponding with the transient appearance of swimming, a rapid but transient efflux of K+ and influx of H+ were observed. With decreases in the rate of H+ influx, the amount of motility decreased. A rate of H+ influx higher than 0.2 mumol/s per ml of cell water gave translational swimming. These results suggest direct coupling of H+ influx to rotation of bacterial flagella.


Partial purification and characterization of thiosulfate oxidase from Pseudomonas aeruginosa. Soluble thiosulfate oxidase from Pseudomonas aeruginosa was purified 85-fold and coverted thiosulfate to tetrathionate by using either ferricyanide or cytochrome c as an electron acceptor.


Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properties,  and comparison with the fucose-induced enzyme. Escherichia coli are capable of growing anaerobically on L-rhamnose as a sole source of carbon and energy and without any exogenous hydrogen acceptor. When grown under such condition, synthesis of a nicotinamide adenine dinucleotide-linked L-lactaldehydepropanediol oxidoreductase is induced. The functioning of this enzyme results in the regeneration of nicotinamide adenine dinucleotide. The enzyme was purified to electrophoretic homogeneity. It has a molecular weight of 76,000, with two subunits that are indistinguishable by electrophoretic mobility. The enzyme reduces L-lactaldehyde to L-1,2-propanediol with reduced nicotinamide adenine dinucleotide as a cofactor. The Km were 0.035 mM L-lactaldehyde and 1.25 mM L-1,2-propanediol, at pH 7.0 and 9.5, respectively. The enzyme acts only on the L-isomers. Strong substrate inhibition was observed with L-1,2-propanediol (above 25 mM) in the dehydrogenase reaction. The enzyme has a pH optimum of 6.5 for the reduction of L-lactaldehyde and of 9.5 for the dehydrogenation of L-1,2-propanediol. The enzyme is, according to the parameters presented in this report, indistinguishable from the propanediol oxidoreductase induced by anaerobic growth on fucose.


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.


Carbon dioxide assimilation in cyanobacteria: regulation of ribulose, 1,5-bisphosphate carboxylase. Cyanobacteria assimilate carbon dioxide through the Calvin cycle and therefore must regulate the activity of ribulose 1,5-bisophosphate carboxylase. Using an in situ assay, as well as measuring the activity in crude, partially purified, and homogeneous preparations, we can show that a number of phosphorylated intermediates exert a regulatory role. Three diverse organisms, Agmenellum quadruplicatum, Aphanocapsa 6714, and Anabaena sp. CA, were studied, and it was found that the in situ and cell-free carboxylase activities were particularly affected by low levels of phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate. There was a marked activation by these ligands when the inactive enzyme was assayed in the presence of low levels of bicarbonate, a result significantly different from a previous report. Moreover, the fully activated enzyme was inhibited by phosphogluconate. In situ Anabaena CA carboxylase activity exhibited a particular capacity for activation by phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate. However, activation of the crude, partially purified, or homogeneous Anabaena CA carboxylase by phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate was significantly decreased when compared with enzyme activity in permeabilized cells. It appears that the microenvironment or the conformation of the enzyme within the cell may be significantly different from that of the isolated enzyme.


Defective F pili and other characteristics of Flac and Hfr Escherichia coli mutants resistant to bacteriophage R17. Mutants resistant to the donor-specific bacteriophage R17 were isolated from Hfr and Flac-containing strains of Escherichia coli K-12. Thirty-five mutants were examined for the presence of F pili by electron microscopy. The pilus morphology was studied, as were the abilities of the cells to retract their pili and to synthesize new pili. Measurements were made of the efficiency of the conjugal deoxyribonucleic acid transfer and of M13 and R17 phage infection. All mutants had noticeable defects in pilus production, structure, or function. Mutants were found which produced unusually long pili, displayed wide variations in the number of pili per cell, and were deficient in pilus retraction and synthesis. Evidence is presented that there may be two pathways of pilus retraction.


Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae. Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.


Lysis of Escherichia coli mutants by lactose. Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.


Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex. Donor deoxyribonucleic acid strands in the eclipse phase of genetic transformation of pnuemococcus (Streptococcus pneumoniae) are purified as a complex with a cf the deoxyribonucleic acid strand in this complex to digestion by nucleases was shown to be 50- to 1,000-fold less than that of uncomplexed single strands of deoxyribonucleic acid. Deoxyribonuclease I, micrococcal nuclease, Neurospora endonuclease, nuclease P1, and the major endogenous nuclease of cell-free extracts were studied. Sensitivity to nuclease attack was not uniform along the deoxyribonucleic acid strand; sequences of strongly protected bases were separated by more sensitive regions. The minimum size of protected fragments was about 70 bases. A complex of protein with the protected deoxyribonucleic acid segments was obtained after partial digestion. The sizes of these complexes, of the protected deoxyribonucleic acid segments, and of the protein subunit released by complete nuclease digestion, are all approximately identical, as determined by gel exclusion chromatography. Deoxyribonucleic acid strands of eclipse complex were also shown to be particularly well protected from attack by the major pneumococcal endonuclease in cell extracts.


Properties and function of the proton-translocating adenosine triphosphatase of Clostridium perfringens. Growth of Clostridium perfringens was inhibited by compounds which dissipate or prevent the formation of electrochemical proton gradients. Membrane vesicles prepared from this organism exhibited Mg2+-dependent adenosine triphosphatase (ATPase) activity sensitive to N,N'-dicyclohexylcarbodiimide. Mg2+-ATPase activity was optimal of 50 degrees C, but no discrete pH optimum was observed. Adenosine triphosphate-dependent quenching of the fluorescence of the weak base quinacrine by everted membrane vesicles suggested that the Mg2+-ATPase is a proton pump capable of generating an electrochemical proton gradient. Adenosine triphosphate-dependent transport of Ca2+ by everted vesicles was sensitive to uncouplers and inhibitors of the Mg2+-ATPase.


Prevalence of tardive dyskinesia.

Prevalence of tardive dyskinesia. Forty-four epidemiologic studies on tardive dyskinesia were evaluated as to whether they provided information on diagnostic criteria, objective scale and assessment, interobserver reliability, period of observation, and specific interhospital coordination. Studies which met these standards were reviewed for data on class of neuroleptic therapy, dose, duration, continuity of treatment, extrapyramidal toxicity, spontaneous dyskinesias, other drugs and treatment modalities, age and sex. A higher prevalence of tardive dyskinesia has been consistently noted in the elderly and in females. No other predisposing factors for tardive dyskinesia have been conclusively demonstrated thus far. Prevalence of tardive dyskinesia is estimated at 24-56% in chronic neuroleptic users.


The effect of antipsychotic drugs on body weight: a retrospective review. Weight gain in schizophrenic patients during chemotherapy was first reported with chlorpromazine. Since then other antipsychotic drugs have exhibited this effect, while some have reduced weight. A retrospective review of 78 schizophrenic patients revealed that thiothixene, fluphenazine, haloperidol, and thioridazine produced a mean weight gain and loxapine a mean weight loss after 12 and 36 weeks of treatment. The ability of an effective antipsychotic drug, such as loxapine, to prevent weight gain or to produce weight loss offers a clinical advantage in the treatment of those schizophrenic patients where weight gain should be a problem.


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.


The alkaline adenosine triphosphatase activity of 30S dynein after modification of the SH groups. Possible involvement of some of the most reactive SH groups. An apparent 'triphasic' alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and susequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively. The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 microM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mM largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again. This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 microM NEM was not stable under the assay conditions (pH 10-10.2 at 25 degrees C), whereas modification with 0.3 mM NEM stabilized 30S dynein against the assay conditions. The possible significance of the 10 microM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.


Purification and properties of biliverdin reductases from pig spleen and rat liver. Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.


Guinea pig liver aromatic aldehyde-ketone reductases identical with 17 beta-hydroxysteroid dehydrogenase isozymes. Two NADPH-dependent aromatic aldehyde-ketone reductases purified from guinea pig liver catalyzed oxidoreduction of 17 beta-hydroxysteroids and 17-ketosteroids. One enzyme efficiently oxidized 5 beta-androstanes and reduced 17-ketosteroids of A/B cis configuration, whereas the other enzyme efficiently oxidized 5 alpha-androstanes and equally reduced both 5 alpha-and 5 beta-androstanes of 17-ketosteroids. However, aromatic aldehydes and ketones, and 3-ketosteroids were irreversibly reduced by the two enzymes. The two enzymes utilized NADP+ or NADPH as cofactor, but little activity with NAD+ or NADH was found. Phosphate ions enhanced the NAD+-dependent dehydrogenase activity and NADH-dependent reductase activity of the two enzymes, whereas the activities with NADP+ and NADPH were not affected. The ratios of the two activities of ketone reduction and 17 beta-hydroxysteroid oxidation of the two enzymes were almost constant during the purification steps after the two enzymes had been separated by DEAE-cellulose chromatography. By kinetic studies and electrophoresis and isoelectric focusing experiments it was confirmed that both of the two enzymes were responsile for the reduction aldehydes, ketones, and ketosteroids and for the oxidation of 17 beta-hydroxysteroids. These results indicate that 17 beta-hydroxysteroid dehydrogenases may play important roles in the metabolism of exogeneous aldehydes and ketones as well as steroids.


The charge stoichiometry of cytochrome c oxidase in the reconstituted system. Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.


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.


Conformation in solution of hemoglobin Osler (alpha 2 A beta 2 145 Tyr replaced by Asp). Computer simulations of Gelin and Karplus ((1977) Proc. Natl. Acad. Sci. U.S.A. 74, 801-805) suggest that in hemoglobin upon ligation the penultimate tyrosyl residues of the subunits are not expelled from the hydrophobic pockets described in the crystals between the helices E and F (Perutz, M.F. (1970) Nature 228, 726-737). This implies that both the liganded and unliganded conformations of hemoglobin may be affected by mutations involving such residues. Investigation of the conformational behavior of liganded and unliganded hemoglobin Osler was conducted measuring the functional properties, the subunits dissociation, the CD and electronic spectra, the protons absorption upon interaction with polyanions, and the reactivity of the -SH groups of the protein. The results suggest that both the liganded and unliganded conformations of the system are affected by the mutation, confirming the anticipations of Gelin and Karplus on the relevance of tyrosine at beta 145 for both allosteric states of hemoglobin.


Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients. The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.


NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin. NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.


Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function. Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.


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.


Nitric oxide induced conformational changes in opossum hemoglobin. Opossum hemoglobin assumes a T quaternary structure upon NO ligation in the absence of organic phophates at pH 6.7. In addition, stripped opossum hemoglobin exhibits a low oxygen affinity when compared to human hemoglobin and a pH-dependent heme-heme interaction with an n value of 2.14 at pH 7.0 and 2.46 at pH 7.35. These observations indicate that opossum hemoglobin may have a destabilized oxy structure when compared to hemoglobin A due to differences in primary structure. Thus, the strong trans ligand effect of nitric oxide is able to disrupt the proximal histidine-iron bond in the alpha-hemes triggering a conformational transition to the T state. Absence of a distal histidine in the alpha-subunits and, therefore an impaired donor acceptor interaction with the sixth ligand, could contribute to the lack of stability of the R quaternary structure in opossum nitrosylhemoglobin. The reduced oxygen affinity of opossum hemoglobin may be compensated for by other physiological factors such as a reduced phosphate effect.


The interaction of inositol pentaphosphate with the hemoglobins of highland and lowland geese. We have studied the binding of inositol pentaphosphate (IPP) to the hemoglobins from two species of goose living at low and high altitudes, using the proton absorption method. Measurements were done at 25 and 37 degrees C in a pH range between 6.0 and 8.8. The bird hemoglobins show a high affinity and a binding stoichiometry of 1 IPP molecule/hemoglobin tetramer both in the ligated and unligated state, indicating the same binding site for IPP in oxy- and deoxyhemoglobin. The results indicate that the interaction of IPP with both geese hemoglobins is very similar. For the deoxyhemoglobins of both species the IPP-binding constant shows a strong pH dependence extending over a wide pH range (i.e. +/- 2 x 10(6) M at pH 8.8 and +/- 6 x 10(10) M at pH 6.0). The binding constant of IPP for the oxyhemoglobins shows a much weaker pH dependence (i.e. +/- 4 x 10(4) M at pH 8.8 and +/- 3 x 10(6) M at pH 6.0), indicating that the interaction of IPP with the goose hemoglobin is strongly dependent on the state of ligation of the protein. The IPP binding constants for the oxy- and deoxyhemoglobins are found to be in good agreement with the IPP-induced change in oxygen affinity of both hemoglobins as estimated from oxygen binding curves.


Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin. Human deoxyhemoglobin has been titrated with nitric oxide at several pH values ranging from 6.0 to 9.0, in the presence and absence of the allosteric effector inositol hexaphosphate at 25 degrees C. Samples were frozen for EPR measurements or analyzed optically within 30 s after mixing to ensure a kinetic population of intermediates. Fractions of pentacoordinate alpha-NO heme groups were determined by fitting EPR and absorbance difference spectra in terms of linear combinations of standard signals. Equivalent results were obtained by these techniques. The fraction of alpha-NO heme exhibiting pentacoordinate character in Hb4NO increases from 0.07 to 0.73 in going from pH 9 to 6. The fraction of alpha hemes which are pentacoordinate in fully saturated nitrosyl hemoglobin, Hb4(NO), increases from 0.0 to 0.41 over the same pH range. Only in the presence of bound inositol-P6 are all 4 the alpha-NO hemes pentacoordinate. Thus, the expression of modified NO heme character is not simply a reflection of the formation of low affinity quaternary conformations. Rather, within this conformation the alpha chain iron atoms exhibit an equilibrium between hexa- and pentacoordinate structures which is perturbed markedly by both proton and phosphate binding. No intermediate coordination structure of the type suggested by Chevion et al. (Chevion, M., Stern, A., Peisach, J., Blumberg, W.E., and Simon, S. (1978) Biochemistry 17, 1745-1750) appears to occur since the observed alpha-NO heme spectra can always by represented quantitatively as a linear combination of the normal hexacoordinate and pentacoordinate signals. The formation of pentacoordinate alpha-NO causes this subunit to exhibit a higher affinity for nitric oxide. Thus on standing at low levels of saturation, there is a slow (t1/2 approximately equal to 8 min at pH 7, 25 degrees C) re-equilibration of ligand from beta to alpha subunits. The final ratio of alpha-NO to beta-NO is 2 to 1 in the absence of phosphates and greater than 10 to 1 in the presence of inositol hexaphosphate.


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.


A calorimetric study of the interaction of ATP with rabbit muscle phosphofructokinase. The heat of interaction of ATP with phosphofructokinase from rabbit muscle was determined at 25 degrees C in 0.1 M potassium phosphate, pH 7.0 and 8.0. The limiting value of the enthalpy change at high ATP concentrations was found to be -11.5 kcal mol of enzyme polypeptide chains. Since phosphate and imidazole have very different heats of ionization (+0.8 and +7.5 kcal/mol, respectively), this suggests that the binding of at least two protons to the enzyme occurs concomitantly with the binding of ATP at the regulatory site.


Effects of thiols, sugars, and proteins on nitric oxide activation of guanylate cyclase. Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.


Fatigue in industry.

Fatigue in industry. Physical fatigue is a painful phenomenon which is localised in overstressed muscles. Mental fatigue is a diffuse sensation of weariness; it is a functional state, one of several intermediate conditions between the two extremes of alarm and sleep. A neurophysiological model of fatigue, involving an activating and inhibitory system has been developed. Fatigue in industrial practice has clinical symptoms: psychic instability, fits of depression and increased liability to illness. Indicators of fatigue are work of performance, subjective feelings of fatigue, electroencephalography, flicker-fusion frequency and various psychomotor and mental tests. Several field studies do, to some extent, confirm the above-mentioned concept of fatigue.


Characterization of anaerobic gram-negative bacilli by using rapid slide tests for beta-lactamase production. A total of 175 isolates of anaerobic gram-negative bacilli were tested for beta-lactamase production by using a slide test modification of the chromogenic cephalosporin (Nitrocefin, Glaxo, Middlesex, England) assay and the iodometric slide test. Included isolates were Bacteroides melaninogenicus (46), B. fragilis (78), other Bacteroides isolates (21), Fusobacterium (25), and other gram-negative bacilli (5). Both slide tests detected 25 B. melaninogenicus isolates that were beta-lactamase producers (minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml). beta-Lactamase produced by the other gram-negative anaerobes could only be detected by the Nitrocefin assay. This assay was positive in 70 or 77 B. fragilis against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml. Ten of 11 other species of Bacteroides against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml were also Nitrocefin test positive. Minimal inhibitory concentrations of penicillin against all isolates of Fusobacterium and unidentifified gram-negative bacilli were less than or equal to 0.78 micrograms/ml and were Nitrocefin assay negative. beta-Lactamase-producing strains of B. melaninogenicus can be differentiated because both the slide iodometric and Nitrocefin assays will be positive, whereas beta-lactamase produced by other Bacteroides will only be detected by the Nitrocefin assay. Such penicillin-resistant isolates could be detected and reported to clinicians before final identification.


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.


Humoral factor that specifically regulates factor X levels in rabbits (coagulopoietin-X). A heat-stable humoral substance (coagulopoietin-X) is present in rabbits partially depleted of Factor X, which is capable of raising Factor X levels when injected into recipient rabbits. Rabbits were partially depleted of Factor X by slow infusion of a globulin fraction of goat anti-rabbit Factor X antibody. This resulted in the reduction of Factor X to 40--50% of normal at 1 h and 60--70% of normal at 6 h. No effect was noted on levels of Factors II, V, or VII. Plasma from these animals, when injected into 10 recipients, specifically raised Factor X levels when measured by four different assay: one-stage assay with bovine VII- and X-deficient plasma and Russell's viper venom; one-stage assay with human X-deficient plasma and thromboplastin; chromogenic substrate assay with Russell's viper venom; and an immunologic assay (Laurell technique). No rise was noted in two control experiments in which normal plasma was injected into recipient rabbits from 2 rabbits injected with a globulin fraction of normal goat serum, nor in 12 rabbits injected with plasma from normal rabbits, nor in 5 rabbits injected with boiled plasma from normal rabbits. The rise in biologic activity of 120--150% of base line was significantly greater than the rise in immunologic activity of 114--117% of base line (P less than 0.05) on 3 different days, suggesting the production of a molecule with greater specific activity rather than increased protein synthesis.


Activation of particulate guanylate cyclase by nitroprusside and MNNG after filipin treatment. Particulate guanylate cyclase from rat lung was stimulated less than 2-fold by agents capable of activating the soluble guanylate cyclase, including sodium nitroprusside, MNNG, azide and hydroxylamine. The action of the first two agents was potentiated by 10 mM 2-mercaptoethanol, and that of the last two by catalase. Pretreatment of the particulate enzyme with the polyene antibiotic, filipin, potentiated the stimulatory effects of the activators, activity with 1 mM nitroprusside in the presence of 2-mercaptoethanol being increased 10.4-fold over basal. The enzyme treated with filipin and nitroprusside showed less specificity for Mn2+, as it was able to use Mg2+ as sole cation more efficiently than the untreated enzyme. Since filipin is known to alter membrane fluidity by interacting with membrane cholesterol, it is proposed that the activity of membrane bound guanylate cylase may be regulated in part by the fluid state of the phospholipid matrix.


Inorganic constituents of milk: I. Correlation of soluble calcium with citrate in bovine milk. A high correlation was found between soluble calcium and citrate concentrations during a study of seasonal changes in the composition of the commercial milk supply to South-west Scotland. A simple physicochemical model of the ion equilibria in the aqueous phase of milk suggests that the observed correlation is causal. This hypothesis was corroborated by an analysis of data on individual cow milks and by experiments involving the addition of citrate to milk at constant pH.


Recognition and management of the depressed dental patient. Depressive illness may affect the patient's response to dental care. It has been implicated both as a causal factor and a sequela in facial pain syndromes. Depression is treated with various medications that may influence function and health of the oral cavity and that may adversely interact with drugs used to control pain and anxiety.


Slow reacting substance (SRS) from ionophore A23187-stimulated human leukemic basophils. I. Evidence for a precursor role of arachidonic acid and initial purification. SRS was generated from human leukemic basophils upon stimulation with ionophore A23187. Radiolabel from [14C]-AA was incorporated into SRS with continued comigration of radioactivity and bioactivity through several chromatographic systems including DEAE-cellulose, silicic acid, and RP-HPLC. Human basophilic leukemia SRS displayed physiochemical properties similar to those of rat basophilic leukemia cell SRS.


A combined alkali extraction--ethidium bromide technique for the measurement of DNA in small pieces of tissue. Alkaline solutions (0.1--0.5 N NaOH) at elevated temperatures can be used to extract DNA from small pieces of tissue. RNA is destroyed by the treatment. In tissues which have been previously exposed to tritiated thymidine, aliquots from the extracting solution can be used directly for the determination of DNA by ethidium bromide fluorescence and radioactivity in both DNA and the nucleotide pool.


Vitamin B12 absorption studied by vascular perfusion of rat intestine. The study of vitamin B12 release from the ileal enterocyte has been hampered by the fact that B12 does not cross the serosa of traditional everted ileal sacs. We studied this release by perfusing the superior mesenteric arteries of starved, heparinized, etherized rats and collecting perfusate from the superior mesenteric vein. The rats were fed 57CoB12 well before study. The standard perfusion medium was Krebs-Henseleit-NaHCO3 buffer containing glucose, dextran, albumin, propranolol, and dexamethasone. The preparation utilized glucose and O2, produced lactate, and was relatively impermeable to [14C]inulin, to D-xylose, and to 57CoB12 bound to an inert human IF. Glucose placed in the gut lumen was transported much more rapidly than D-xylose. Vitamin B12 emerged in the perfusate bound to a protein with a molecular size similar to that of TC II. IF could not be identified in the perfusate. Rat serum, independently of its unsaturated TC II content, increased the rate of transfer of B12 into the perfusate.


Characteristics of azurin from Pseudomonas aeruginosa via 270-MHz 1H nuclear magnetic resonance spectroscopy. Assignments of resonances in the 1H nmr spectra of Cu(I) azurin to proton groups in the protein are discussed in detail. Comparisons are drawn between Cu(I), Cu(II), apo, Hg(II), and Co(II) azurin samples. Redox titration of Cu(I) azurin with K3Fe(CN)6, is used to correlate Cu(I) and Cu(II) 1H nmr spectral features, and observed line broadenings deriving from Cu(II) paramagnetic effects are used to deduce the distances of assigned proton groups from the copper center. Histidine residues are characterized in terms of pK values, rates of acid-base exchange near the the pK, and rates of C2H exchange with solvent deuterium. The possibility of histidine involvement in the azurincytochrome 551 electron exchange mechanism is discussed. A small number of NH protons observed to be distinctively inert to 2H exchange with solvent 2H2O, in the Cu(I) protein, are found to show increased lability on removal of the metal.


Influence of cerebral embolism on brain monoamines. In baboons the right cerebral hemisphere was embolised by a shower of microemboli, immediately followed by one large embolus designed to occlude the middle cerebral artery (MCA). One hour after embolism a significant, though small, reduction in blood flow and oxygen consumption of the embolised hemisphere was recorded, at which time the animals were killed and brain monoamines measured. Dopamine was reduced in the ipsilateral caudate nucleus, the reported site of maximal ischaemic damage in this model. Dopamine levels were increased in frontal and occipital grey matter sampled from areas surrounding the occluded MCA territory and in similar brain areas of the opposite non-embolised hemisphere. Noradrenaline was increased in grey matter from both cerebral hemispheres, as well as subcortical structures bilaterally. Brain 5-hydroxytryptamine levels were unaltered, but increased 5-hydroxyindoleacetic acid in cisternal cerebrospinal fluid suggested transient alteration in 5-hydroxytryptamine metabolism after embolism. The effects of cerebral embolism on brain monoamine metabolism appear to be different from the effects of permanent surgical occlusion of major cerebral vessels. The bilaterality of effects after unilateral hemispheric embolism might be related to diaschisis. The mechanisms of the observed changes, as well as their relevance to the progression of cerebral ischaemia and the complications associated with cerebral embolism, still require to be established.


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.


Purification and properties of transketolase from pig liver. I. An attempt to resolve the enzyme into apoenzyme and cofactors. Transketolase, sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase [EC 2.2.1.1], was extracted from pig liver and purified 96-fold by ammonium sulfate fractionation, followed by column chromatography using DEAE-cellulose and a Sephadex G-200. Transketolase from pig liver was stable at pH 6.0 and above, whereas it was unstable at lower pH values. It could be resolved into apoenzyme and thiamine pyrophosphate in an acidic medium, in contrast to baker's or brewer's yeast transketolase which resolved in an alkaline solution. All the activity of pig liver transketolase was lost upon incubation at pH 5.0 for two hours even at 0 degrees C but about 40% of the original activity could be restored by the addition of excess thiamine pyrophosphate and CaCl2. Restoration of the activity was achieved effectively at pH 7.6-8.0.


Influences of oyster or clam feeding on lipid metabolism in rats. Rats were fed on three kinds of diets for two weeks: (I) basal diet, (II) containing 0.1% cholate and (III) containing 0.1% cholesterol and 0.1% cholate. Each dietary group was further divided into subgroups to whose diet was added 0, 5 or 10% (dry weight) of minced oyster (Callocorchina) or clam (Tapes japonica). The serum and liver cholesterol levels of the rats fed the basal diet were reduced by feeding oyster or clam. The serum and liver triglyceride levels of all dietary groups were lowered markedly by feeding oyster or clam. The activities of glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase were markedly reduced in the basal groups fed oyster or clam. These effects were observed in 5 and 10% shellfish feeding. These shellfish may be considered hypolipidemic foods.


Fluoride action on acid resistance of unaltered human surface enamel. Unaltered surface enamel of extracted human teeth was subjected to tests of resistance to dissolution in 10 mM acetic acid at pH 4.0 and 10 mM EDTA at pH 7.4 in a miniature continuous flow system. Fluoride in EDTA had no effect on enamel dissolution. Inclusion of 0.25, 0.50 and 5.0 mM NaF in the acid at pH 4.0 greatly increased the resistance of the enamel to dissolution. The 0.5 mM NaF in the acid almost instantly reduced enamel dissolution and the effect persisted through the 1 h experiments. These results suggested that the acid labile fluoride in plaque and tooth minerals may provide the primary beneficial effect of all types of enamel fluoridation.


An in vitro model for assessing drug availability from lipophilic vehicles. An apparatus for studying the rate of release of a solute from a water-immiscible solvent into an acidic, aqueous liquid, followed by permeation through a simulated lipid membrane, is described. The object was to imitate the absorption of a drug after oral administration in a soft gelatin capsule. Rate constants of transfer were determined for a series of substituted benzoic acids, using octanol and isopropyl myristate as solvents. The quantity of solute in the acidic phase did not correlate with the solvent-water distribution coefficient, but was linearly related to the rate constant for transfer from solvent to water. A dynamic system was therefore postulated, rather than one in which the two phases are in equilibrium. In vivo studies on two of the solutes confirmed the in vitro observations. No simple relationship could be derived between blood concentrations and any in vitro parameter, but the rank order of magnitude of the blood concentrations fitted the postulated dynamic mechanism.


Precise spectrophotometry of small quantities of drugs in the presence of irreproducible quantities of impurities. A variety of methods of spectrophotometric assay of compounds in the presence of one or more irrelevant impurities have been recently described, employing, for example, the use of Legendre polynomials and trigonometric functions. The use of least-squares solutions of overdetermined systems (Scheid 1968) in spectrophotometry is outlined, with conditions under which simplified overdetermined systems have been used for precise spectrophotometric assay of small quantities of drug substances in the presence of largely irreproducible quantities of irrelevant impurities.


The effects of physiocochemical properties of pethidine and its basic metabolites on their buccal absorption and renal elimination. The pKa values, RF values, partition coefficients between n-heptane and phosphate buffer (pH = 7.4), and buccal absorption tests of pethidine and its basic metabolites, norpethidine and pethidine N-oxide, have been determined. The amounts of these compounds recovered in the 48 h urine samples after intramuscular administration of pethidine (1.5 mg kg-1) to six healthy subjects varies with urinary pH values: the recovery from acidic urine (pH 5.0) is 15.2 to 52.0%, 6.7 to 12.9% and 0.2 to 2.3% of dose for pethidine, norpethidine and pethidine N-oxide respectively; in alkaline urine (pH 8.0) the values are 0.8 to 1.8%, 0.6 to 2.8% and 0.3 to 2.1% respectively. The physicochemical properties (pKa values, RF values, partition coefficients) and buccal absorption of pethidine, norpethidine and pethidine N-oxide are in good agreement with the pattern of their renal elimination in acidic and alkaline urine conditions. The contribution of the physiocochemical properties of pethidine and its metabolites to the drug's disposition in the body and the effect of urinary pH on its metabolism should be taken into account in pharmacokinetic studies and interpretation of intersubject variation in response to pethidine.


pA2 values of selective beta-adrenoceptor antagonists on isolated atria demonstrate a species difference in the beta-adrenoceptor populations mediating chronotropic responses in cat and guinea-pig. pA2 values for atenolol (beta 1-selective) and alpha-methylpropranolol (beta 2-selective) have been determined on isolated atria of cat and guinea-pig using noradrenaline (beta 1-selective) and fenoterol (beta 2-selective) as agonists. On guinea-pig atria, the pA2 values did not vary with the agonist used. On cat atria the pA2 for atenolol was greater with noradrenaline than with fenoterol and the pA2 for alpha-methylpropranolol was greater with fenoterol than with noradrenaline. Fenoterol was 20 times more potent on cat than on guinea-pig atria whereas noradrenaline was approximately equipotent in the two species. The results have been intrepreted as suggsesting that both cat and guinea-pig atria contain one receptor type in common (beta 1) but that only cat atria contain beta 2-adrenoceptors as well.


Role of the dopaminergic system in the cataleptogenic action of bulbocapnine. Time courses of the behavioural and biochemical effects of a cataleptogenic dose (50 mg kg-1 i.p.) of bulbocapnine have been studied in the rat. Catalepsy ensues immediately after administration of the drug and lasts 1 h. Concomitant with the onset of catalepsy there is a rise in HVA and DOPAC concentrations in whole and discrete parts of the brain (striatum, limbic system). Dopamine content does not change in whole brain but it decreases in the striatum and increases in the cortex and hippocampus. No significant effects on NA, 5-HT and 5 HIAA concentrations were observed.


Cardiovascular studies with impromidine (SK&F 92676), a new very potent and specific histamine H2-receptor agonist. Impromidine (SK & F 92676) has recently been identified as a potent and specific histamine H2-receptor agonist. The present paper describes some cardiovascular studies with the drug. Impromidine lowers blood pressure in cats and rats by interaction with H2-receptors. During continuous intravenous infusions of impromidine, the fall in blood pressure is due to a reduction in total peripheral resistance; cardiac output increases during hypotension. The responses to impromidine are similar to responses to histamine in mepyramine-treated cats. Impromidine administered intra-arterially also causes vasodilatation in the femoral and mesenteric vasculature by interaction with H2-receptors. Impromidine stimulates all measured parameters including coronary flow in the isolated working heart of the guinea-pig. Dose-response curves to impromidine were displaced to the right in the presence of cimetidine.


Haemodynamic effects of the carboxylic ionophore monensin when administered before and during shock induced by E. coli endotoxin. The haemodynamic effects of the carboxylic ionophore monensin have been examined in cats anaesthetized with sodium pentobarbitone. Marked increases in left ventricular dP/dtmax (and dP/dt at fixed isovolumic pressures) and slight increases in cardiac output and stroke volume occurred, indicating increased myocardial contractility. Heart rate was unchanged but systemic arterial pressure was substantially increased. Satisfactory increases in contractility and arterial pressure were obtained when monensin was infused intravenously in a total dose of 0.25 mg kg-1 over 10 min. Larger doses, especially if rapidly injected, resulted in very marked increases in myocardial contractility leading eventually to cardiac failure. The haemodynamic effects of monensin were markedly reduced during shock induced by E. coli endotoxin and there was unfortunately no evidence to suggest that this extremely potent compound might be potentially beneficial in this form of profound cardiovascular shock.


Anti-inflammatory and platelet anti-aggregant activity of phospholipase-A2 inhibitors. Mepacrine, papaverine, p-bromophenacyl bromide and 2,3-dibromo(4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid (CB 874) inhibit the hydrolysis of phospholipids induced by thrombin in dog platelets. They also exhibit anti-inflammatory and anti-aggregant properties. These biological activities may be explained by a direct or indirect inhibitory action on phospholipase A2. Phospholipase A2 inhibitors may block not only the release of arachidonic acid and its subsequent conversion into prostaglandins but also the formation of lysophospholipids involved in inflammation and/or platelet aggregation.


Non-competitive-non-equilibrium alpha-adrenoceptor blocking properties of N-benzyl iodoacetamide, betsamide. N-Benzyl iodoacetamide, betsamide, at 10 mg kg-1 i.v. blocked the hypertensive and contractile responses of the nictitating membrane of the cat to adrenaline. The blockade had a lag period before full development. Pretreatment of cats with betsamide for 7 or 18 h showed a non-equilibrium type of alpha-adrenoceptor blockade. The responses of the nictitating membrane to adrenaline were markedly depressed and did not recover after high doses of adrenaline. In the same cats, adrenaline caused a profound hypotension. The effect of betsamide lasted for at least 72 h. In the rat isolated vas deferens, 3 X 10(-5) M betsamide non-competitively blocked the contractile responses to noradrenaline; the adrenoceptor blockade was less effective when betsamide was applied with noradrenaline. The blockade lasted for more than 24 h, and was not reversible after extensive washing. Betsamide antagonized the contractile effects of carbachol and 5-hydroxytryptamine on the rat vas deferens, but not the beta-responses of the guinea-pig trachea to adrenaline and isoproenaline. Results are discussed in relation to a probable mechanism of action.


Potentiation of the diabetogenic effect of streptozocin by phentolamine in the rat. The effect of alpha- and beta-adrenoceptor blocking agents and a beta2-agonist, salbutamol, on the diabetogenic effects of streptozocin (threshold doses) was investigated in the rat. Phentolamine and salbutamol potentiated the diabetogenic effect of streptozocin but phenoxybenzamine, tolazoline, oxprenolol and propranolol were without effect. The potentiating effect of phentolamine was blocked by oxprenolol. Potentiation of the diabetogenic effect by phentolamine is not related to alpha-adrenoceptor block, vasodilatation or insulin release. But it may be related to its ability to stimulate beta-adrenoceptors.


Effect of 5-hydroxytryptamine on blood glucose and cyclic AMP in the rat. The effects of 5-hydroxytryptamine (5-HT) on plasma cyclic AMP (cAMP) and glucose concentrations were studied in rats in vivo. 5-HT injected i.p. increased plasma cAMP and glucose. Injections of propranolol, hexamethonium, and cyproheptadine inhibited the 5-HT-induced increase in glucose but not in cAMP. Atropine did not inhibit the action of 5-HT. These effects of 5-HT were not seen in adrenomedullectomized rats, and 5-HT did not elevate the concentration of plasma cAMP in anti-glucagon antiserum-injected rats. These results confirm the previously reported finding that 5-HT-induced increase in blood glucose is mediated via adrenaline released from adrenal medulla by 5-HT and suggest that the increase in plasma cAMP, induced by 5-HT, is due to glucagon released by an unknown factor, or factors other than adrenaline released from the adrenal medulla by 5-HT.


The rapid development of functional tolerance to ethanol by mice. A method is described in which the development of tolerance to ethanol in individual mice can be measured during the inhalation of ethanol vapour. This method has been used with two behavioural end-points, loss of righting reflex and loss of rotarod performance. It demonstrates that, in the adult male, TO Swiss mouse, peak tolerance, in which approximately 2 X the original effective blood ethanol concentration is required to produce the behavioural end-point, can develop in 3--5 h. After this time the ability of the animals to perform normally in the presence of continued high concentrations of ethanol in blood begins to fall. The results are discussed in relation to current concepts of tolerance to central nervous system depressant drugs.


Aspirin-caffeine interaction in the rat. Both aspirin at a high dose (400 mg kg-1) and caffeine (5 mg kg-1) induced hyperactivity in the DA rat, but lower doses of aspirin were without effect. Caffeine-induced hyperactivity was brief (2 h) but that due to aspirin was evident from 1--6 h after dosing. Co-administration of the two drugs caused long-lasting hyperactivity, even with doses of aspirin which had no stimulant effects themselves. Absorptive and metabolic effects did not appear to play a major role in the interaction. The most likely effect is that of salicylate on catecholamine utilization in the central nervous system, which is compounded in the presence of a phosphodiesterase inhibitor.


The effects of some sweetening agents and osmotic pressure on the intestinal absorption of sulfafurazole in the rat. The effects of some sugars and osmotic pressure have been examined on the absorption of sulfafurazole in the rat. Statistically significant correlations were found among the hypertonic solutions between the reciprocal of osmotic pressure and AUC0--infinity or Cmax. In addition, negative correlation existed between plasma concentrations at 15 min and osmotic pressure. No significant correlations were noted among the hypotonic solutions. More attention should be paid to the osmotic pressure of formulations. The osmotic pressure of hypertonic solutions, especially, might affect absorption of drug from its dosage form.


The use of phospholipid liposomes for lithium administration. Polydipsic effect and tissue distribution of lithium. Administration of lithium entrapped in phospholipid liposomes increased the lithium-induced polydipsia, but did not accelerate the onset of this effect. It also resulted in a larger accumulation of lithium in the liver, kidney and spleen, but not in the brain. The time course of polydipsia suggests that it depends on the intracellular lithium concentration. However, the rate of development of this effect depends on some additional factor.


Dissolution of bupivacaine 3-hydroxy-2-naphthoate into phosphate buffers. Dissolution behaviour of bupivacaine 3-hydroxy-2-naphthoate (BUPNH) into phosphate buffers, pH 7.4 was investigated at 25 degrees and 37 degrees C. Although the dissolution pattern of the salt at 25 degrees C was normal, i.e., the concentrations of the acid and base component agreed, during the entire dissolution period, that at 37 degrees C was anomalous. The dissolution consisted of two phases, i.e. an initial normal phase was followed by a second slower phase in which the concentration of the acid became, at equilibrium, markedly higher than that of the base. This unusual dissolution behaviour at 37 degrees C was shown to be due to the precipitation of the base, which, in turn, is attributed to the unusual temperature dependency of the solubility of the base in phosphate buffers, i.e. decrease in solubility with increasing temperature.


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.


Sensitive fluorescence assay for d,l-methadone. d,l-Methadone forms a fluorophore when reacted with paraformaldehyde in concentrated sulfuric acid. Based on this reaction, a fluorescence assay suitable for quantitative d,l-methadone analysis from plasma and other tissues was developed. d,l-Methadone was extracted at pH 9.2 from the deproteinized filtrate of plasma or of aqueous tissue homogenate into an organic phase of 25% isobutanol in ethylene dichloride. After an aliquot of the organic phase was evaporated to dryness at 50--55 degrees with an air jet, paraformaldehyde (0.1%, in concentrated sulfuric acid) was added, and fluorescence was read at 450 nm with excitation at 275 nm. By this method, d,l-methadone can be assayed in the presence of its metabolites, morphine, diacetylmorphine (heroin), codeine, and cocaine; however, amphetamine, meperidine, and quinine interfere.


Dissolution rates of doxycycline free base and hydrochloride salts. The dissolution rates of doxycycline monohydrate, hyclate, and hydrochloride dihydrate crystal forms were investigated using the static pellet method. Solubility product equilibria with chloride ion strongly suppressed the dissolution rate of the hydrochloride dihydrate salt. This form dissolved about fourfold slower in 0.1 N HCl than in water, which was consistent with its solubility in these media. Specificity for chloride was demonstrated by the rapid dissolution rate for the hydrochloride dihydrate in 0.1 N methanesulfonic acid. The dissolution rates of the hyclate, a solvated hydrochloride salt, and the free base were not sensitive to chloride ion. The results show that common ion equilibria with chloride can strongly reduce the dissolution rate of a thermodynamically stable hydrochloride salt form, while the free base or a metastable hydrochloride salt are not similarly affected.


Investigation of narcotics and antitussives using drug discrimination techniques. Rats learned drug discriminations in a shock-escape T-maze task. They were trained to turn right in the maze following injection of a drug (D) and left when no injection (N) was given. Number of training sessions before criterion performance (STC) was used to indicate degree of discriminability of the training drug. STC decreased monotonically as dosage increased, and reached a minimum of 3 to 26 with various agonists. Most agonists were not highly discriminable. Daily maintenance injections of morphine, 200 to 600 mg/kg, increased the STC of morphine, 15 mg/kg, significantly, but complete tolerance to discriminable drug actions was not observed. After rats discriminated D vs. N, they were tested with novel drugs to determine which would elicit D choices. Most morphine-like agonists substituted for one another during substitution tests; the tested agonists included alphaprodine, codeine, fentanyl, heroin, meperidine, methadone, morphine, piminodine and propoxyphene. In a few instances, one of these agonists failed to substitute for another. Naloxone and naltrexone antagonized the discriminable effects of morphine. Cyclazocine, levallorphan, naltrexone, dextromethorphan, ethoheptazine and the narcotic agonists did not substitute for one another, suggesting that six dissimilar discriminable effects were produced by these drugs.


Stimulation by cyclic GMP of sodium efflux in barnacle muscle fibres. 1. The ouabain-insensitive Na efflux in barnacle muscle fibres is promptly stimulated by injection of cyclic GMP. The minimal effective injected concentration is found to be about 10(-7) M. This effect of cyclic GMP could not be mimicked by injecting 5'-GMP. 2. External application of ouabain (10(-4) M) to fibres not pretreated with ouabain during the stimulatory response to cyclic GMP causes some inhibition of the Na efflux indicating that cyclic GMP does not cause appreciable inhibition of the Na:K pump. 3. The magnitude of the stimulatory response to injected cyclic GMP depends on the external Ca2+ concentration, as well as pHe but not on the Na+, K+ or Mg2+ concentration. It also depends on pHi, since acidification of HCO3-containing ASW leads to a greater enhancement of the response to cyclic GMP than is observed with acidified HERPES-ASW. 4. Stabilization of myoplasmic pCa by injecting 100 mM-EGTA before or after cyclic GMP fails to alter the magnitude of the response to the nucleotide. Enrichment of the fibre with Mg2+ at the time of injection of cyclic GMP leads to a reduced response. No change in response, however, is seen when the internal free Mg concentration is suddenly reduced by injecting 0.05 M-pyrophosphate with cyclic GMP. 5. Injection of cyclic GMP-dependent protein kinase stimulatory modulator before cyclic GMP fails to enhance the response to the nucleotide. The same is true of the phosphodiesterase inhibitor protein. However pre-injection of 10(-2) M-papaverine enhances the response to a subsequent injection of 10(-3) M-cyclic GMP. 6. Injection of pure protein kinase inhibitor (1.6 x 10(-4) M) before 10(-3) M-cyclic GMP reduces the response to the nucleotide. 7. The argument is put forward that injected cyclic GMP stimulates the ouabain-insensitive Na efflux mainly by activating cyclic AMP-protein kinase rather than cyclic GMP-proton kinase.


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.


The nature of the negative endocochlear potentials produced by anoxia and ethacrynic acid in the rat and guinea-pig. 1. The alterations in the Na+ and K+ concentrations of the cochlear endolymph and in the endocochlear potential were followed simultaneously by means of ion-sensitive and conventional micro-electrodes during simple anoxia, during anoxia after i.v. ethacrynic acid and after i.v. ethacrynic acid alone. The endolymphatic pH changes were measured separately and the effect of perilymphatic ethacrynic acid upon the endocochlear potential was investigated. 2. The over-all Na+:K+ permeability ratio for the endolymph system was determined in individual animals for the first time using an indirect method. The normal mean values of 0.27 (rat) and 0.38 (guinea-pig) were increased after ethacrynic acid. Permeability changes occurred during anoxia but were delayed in onset. 3. The negative endocochlear potentials in each situation behaved quantitatively like modified K+ diffusion potentials largely dependent upon the K+ and Na+ gradients between endolymph and perilymph.


Selective response of rat peripheral sympathetic nervous system to various stimuli. 1. We utilized the induction of tyrosine hydroxylase, a catecholamine-synthesizing enzyme, in sympathetic ganglia and adrenal medullae to explore the central and peripheral mechanisms through which choline, various environmental stresses, and drugs that alter blood pressure or central neurotransmission affect various portions of the sympathetic nervous system. Animals received each treatment chronically, and enzyme activity was measured in the superior cervical, stellate, and coeliac ganglia and in the adrenal medullae.2. Choline administration increased tyrosine hydroxylase activity in all four tissues, probably by increasing the release of acetylcholine from preganglionic sympathetic neurones that synapse on catecholamine-producing ganglion and chromaffin cells; carbachol and nicotine had similar effects.3. Insulin enhanced tyrosine hydroxylase activity primarily in the coeliac ganglion and the adrenal medullae, but not in the superior cervical ganglia.4. Reserpine and phenoxybenzamine increased the activity of the enzyme in all four tissues.5. Prolonged exposure to a cold environment increased enzyme activity in all four tissues, but especially in the stellate and coeliac ganglia; forced swimming affected tyrosine hydroxylase only in these two ganglia.6. Several drugs known to modify central neurotransmission were found to increase tyrosine hydroxylase activity in some portions of the sympathetic nervous system but not in others. 5,7-Dihydroxytryptamine, which destroys terminals of serotoninergic neurones, enhanced enzyme activity in all four tissues, but primarily in the coeliac ganglion and adrenal medullae. ET-495 (a dopaminergic agonist), D-amphetamine, and morphine induced tyrosine hydroxylase activity in the adrenal medullae and the coeliac ganglion, but not in the superior cervical ganglia. Oxotremorine, a centrally acting muscarinic agonist, increased tyrosine hydroxylase activity only in the adrenal medullae; its effect was not blocked by methylatropine, a peripheral muscarinic blocker.7. These data indicate that specific neurones in the central nervous system, which utilize specific neurotransmitters and which are differentially affected by drugs and environmental inputs, selectively influence the outflows through the various zones of the sympathetic nervous system.


HCO3-/Cl- exchange across the human erythrocyte membrane: effects of pH and temperature. Changes in extracellular pH (PH0) in red cell suspensions were monitored in a stopped-flow rapid reaction apparatus under conditions where dpH0/dt was determined by the rate of HCO3-/Cl- exchange across the membrane. Experiments were performed at 5 degrees C less than T less than 40 degrees C using either untreated cells or cells exposed to 0.11 mM SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). Although SITS exposure reduced the rate of exchange by 90%, both untreated and SITS-treated cells are similarly affected by changes in pH0 and temperature. The rate of HCO3-/Cl- exchange exhibits a minimum at about pH0 5 and a maximum at about pH0 7.4 at all temperatures. A transition temperature of 17 degrees C was observed in the Arrhenius relationship for all pH0. The activation energies (Ea) in kcal/mol are 19.6 below and 11.7 above 17 degrees C for 5 less than pH0 less than 8. These findings, similar to those reported for Cl- self-exchange, suggest that: (i) a change in the rate-limiting step for HCO3-/Cl- exchange occurs at 17 degrees C, possibly due to an altered interaction between the transport pathway and membrane lipids; (ii) the carrier system can be titrated by either H+ or SITS from the outside of the membrane, but the untitrated sites continue to transport normally; (iii) the pH0 dependence of the rate of exchange is consistent with the titratable carrier having its most alkaline pK in the range expected for amino groups; and (iv) below pH0 5, the nature of the exchange is markedly altered.


Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 Dalton range. Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1--2 mM each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large and small. (Electrical coupling, however, persisted in a proportion of the junctions tested.) Treatment with the ionophore A23187 (2--10 micrometers) or with CO2 (an atmosphere of 100% CO2 equilibrated with the medium) produced selective junctional blockade: transmission of a 688 and an 817-dalton tracer was generally blocked, while that of a 376-dalton traced and, in certain conditions, that of a 559-dalton one, persisted. The junctional effect of the ionophore required the presence of Ca in the external medium; and effective junctional blockade by CO2 required pretreatment in medium with high Ca concentration or, interchangeably, pretreatment in medium with high CO2 concentration. In one cell type, prolonged exposure to medium with high Ca concentration alone sufficed to block transmission of the 688-dalton tracer. These effects are discussed in terms of the Ca hypothesis of junctional permeability regulation. In comparison with mammalian (or other vertebrate and invertebrate) organized tissues or with insect cell cultures, the mammalian cell cultures are more resistant to junctional blockade. This difference in transmission stability is discussed in terms of the electron-microscopic finding in the mammalian cultures of fine, bilateral cell processes connected by gap junctions.


Inhibitory actions of mercury compounds against glucose-6-phosphate dehydrogenase from yeast. Kinetic studies on the inhibition of the activity of glucose-6-phosphate dehydrogenase with mercuric chloride (MC) and methylmercuric chloride (MMC) have revealed that MC inhibited the enzyme non-competitively, while MMC inhibited it competitively. The Km value was 5.26 X 10(-5) M for glucose-6-phosphate and Ki value of MC was 2.17 X 10(-5) M, while that of MMC was 4.35 X 10(-3) M. The strong complex formation of nicotinamide adenine dinucleotide phosphate (NADP) or amino acids (cysteine, cystine, histidine, tryptophan or tyrosine) with MC was demonstrated in the presence of phosphate buffer as compared with that of MMC in the same buffer.


Developmental anomalies associated with hypospadias. We reviewed 272 patients with hypospadias who were treated surgically to survey the complicated anomalies in these patients. Urogenital anomalies were recognized in 40 per cent of the 272 cases and extra-urogenital anomalies were noted in 9.2 per cent. Undescended testis was the most frequently seen anomaly, having been noted in 13.2 per cent of the 272 cases. The incidence of male vagina or utriculus masculinus was 11.8 per cent, prepenile scrotum 7.7 per cent and hypoplasia of the testis 6.6 per cent. Each of these 4 anomalies associated with hypospadias increased in incidence in proportion to the severity of the penile deformity. The necessity of systemic urological and hormonal examinations was emphasized, especially in cases of severe hypospadias.


An investigation of arterial disease in Alaskan reindeer and Caribou. No significant lesions of atherosclerosis or other vascular diseases were found in the aorta and coronary arteries of 34 reindeer and 15 caribou (Rangifer tarandus). Serum lipid, phospholipid, cholesterol and triglyceride levels were similar in caribou and reindeer and did not differ greatly from those reported in other ruminants.