The formation of mono-N-acetylhexosamine derivatives of dolichol diphosphate by pig liver microsomal fractions.

Incubation of pig liver microsomal preparations with UDP-N[U-14C]acetylglucosamine yields a 14C-labelled lipid. The requirement for Mn2+, the pH optimum, time-dependence and the reversibility by UMP of the transferase are reported. Evidence is presented in favour of the lipid being a mixture of dolichol diphosphate N-[14C]acetylglucosamine and dolichol diphosphate N-[14C]acetylmannosamine. Available data suggest that the epimerization takes place while the hexosamine is bound in this lipid-soluble form. The N-acetylmannosamine appeared not be be released into the medium. The subfractionation of the microsomal fraction to separate transferase activity from membrane-bound beta-N-acetylglucosaminidase activity is also reported.


[Effect of reserpine on the potency of alpha receptor blockers].

The influence of rather low doses of reserpine (0.125--2.0 mg/kg) on the pressor effect of noradrenaline (NA) was studied in pithed rats. In addition it was investigated whether the blocking activity of alpha-sympathicolytic agents is influenced by pretreatment with reserpine. 24 h after injection of these doses of reserpine this drug only increased the pressor effect of lower doses of NA without shifting the dose-response curve to the left. As a rule in rats pretreated with reserpine the affinity of alpha-blocking agents (derivatives of phenylethylamine and phenoxyethylamine) to alpha-adrenoceptors is not considerably altered. Only two phenylethylamine derivates substituted with a p-hydroxy group possessed considerably higher blocking activity in reserpine pretreated animals than in rats not pretreated. The increase in sensitivity of blood vessels against low doses of NA (250 pg and 1 ng) observed in rats pretreated with reserpine is abolished even by alpha-blocking substances with low affinity to alpha-adrenoceptors.


[Bromine concentration in urine following uptake of therapeutic and suicidal doses of carbromal].

The bromine concentration in urine after ingestion of therapeutic and suicidal doses of bromodiethylacetylcarbamide (carbromal) is described. Connections with the renal elimination of sodium and chlorine are described and discussed.


Identification of neutral proteases in human neutrophil granules that degrade articular cartilage proteoglycan.

Human polymorphonuclear neutrophil (PMN) granule extract (25 mug of protein) released 60 percent of the available 35SO4 from labeled rabbit articular cartilage in 0.5 hour at neutral pH. N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanine choloromethyl ketone (NAcAAPACK), a specific elastase inhibitor, was only minimally effective against whole granule extract, and N-alpha-tosyl-L-lysine chloromethyl ketone, which inhibits trypsin but not elastase, was completely ineffective. Preparative disc-gel electrophoresis of PMN granule extract revealed two separate regions with independent activity against 35SO4-labeled cartilage. One region contained elastases and when tested alone, was completely inhibited by NAcAAPACK. The other contained lysozyme and two esterases active against N-acetyl-L-phenylalanine-alpha-naphthol. Purified lysozyme proved inactive, suggesting that the chymotrypsin-like esterases were responsible for proteoglycan degradation by this region of the gel.


[Model for the continuous determination of cell metabolism].

Using the isolated heart of guinea pigs according to the method of Langendorff a new model to study the metabolism of the myocardium is described. In one modification the heart is perfused by continuous flow of Ringer-Locke-solution, and the effluent is discarded. In the other modification the effluent is used for recirculation after saturation with oxygen. The advantage of this method is the possibility to measure oxygen consumption and production of acid metabolites by using pH-electrodes in the perfusion medium before and behind the heart. For measurement, the blood micro system and the acid-base analyzer is used. Many drugs and several unknown compounds have been examined for their influence on heart metabolism and could be classified as either stimulatory, inhibitory or ineffective. It is possible to study dose-response curves of these compounds to compare various compounds and to examine the cumulative or duration effects of combinations. The site of action of a compound and thus the mode of action can be examined as was demonstrated for alpha- and beta-blockers. Perfusion of the heart with glucose-free solution permits measurement of the effect of various energy sources for intensity and duration of effect. The well-known capacity of the heart to use lipids as an energy source could be demonstrated. When a pharmacologically active compound is added to the perfusion medium, glucose consumption, production of lactic acid and of other metabolites can be determined. The results obtained in the isolated heart were confirmed by corresponding tests in the intact animal. The model is highly sensitive to several classes of drugs. In some instances amounts of less than 1 mug can be determined.


The synthesis and pharmacology of a novel benzodiazepine derivative, 1-(beta-methylsulfonylethyl)-5-(o-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4-benzodiazepin-2-one (ID-622).

The pharmacological profiles of a new benzodiazepine derivative, 1-(beta-methylsulfonylethyl)-5-(o-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4-benzodiazepin-2-one (ID-622), were shown. In anti-convulsant test, ID-622 was more potent than diazepam and medazepam when tested with pentylenetetrazol or bemegride, but was less potent than diazepam tested with strychnine or MES in mice. Taming effects of ID-622 were more potent than diazepam in both electroshock- and isolation-induced fighting mice tests, but were less potent in both septal rats and O.B. rats tests. ID-622 had only a weak influence on the spontaneous locomotor activity, and did not cause the righting reflex loss. In EEG, ID-622 increased fast activity and depressed hippocampal I-waves and amygdala after-discharge in cats. Acute toxicity of ID-622 was very low.


Chemico-pharmacological studies on saponins of Panax ginseng C. A. Meyer. II. Pharmacological part.

The pharmacological properties of seven pure saponins isolated from Panax ginseng C. A. Meyer were studied. 1. The ginseng saponins showed weak toxicities in mice. Especially, Rg1, Rf and Rb 1, which contained glucose as a sugar component, were weaker in their toxicities than the rest, which contained arabinose and/or rhamnose. It was also noted that the saponins containing protopanaxadiol as sapogenin were more toxic than those containing protopanaxatriol. 2. All the saponins diminished ACh-induced contraction of the isolated ileum of the guinea pig. On the other hand high concentrations of Rb 2 caused contraction of the ileum by itself. 3. All of the saponins induced a decrease in heart rate and showed biphasic actions on the blood pressure in rats, while they little affected respiration. They caused blood pressure fall preceded by slight rise. Among them, Rg 1 showed the most prominent action and it produced a blood pressure rise with doses of 30 to 100 mg/kg. The pressor as well as depressor action was not influenced by the pretreatment with any of atropine, diphenhydramine, phentolamine and propranolol. 4. Rg 1 and Re showed vasodilator action in dogs, the potencies of which were 1/20 and 1/50 of that of papaverine, respectively. Rc and Rb 2 showed very weak vasodilator actions but Rb 1 did not. 5. Among the 7 saponins, Rd, Re and Rb 2 showed more potent hemolytic actions than those of the rest and the potencies were proportional to their toxicities. 6. Whereas single administration of Rf, Re and Rd significantly suppressed the conditioned avoidance response, repeated administration of them caused facilitation of the response. On the other hand, Rb 2 always showed very weak suppressant action. 7. Rg 1, Rf, Re and Rd significantly suppressed the fighting of mice induced by foot shock, while Rb 1, Rb 2 and Re little affected the fighting. 8. All the saponins showed antifatigue action. They markedly increased the movement after compulsory gait and the action was consistent and independent of their action on the movement before compulsory gait. 9. The saponins showed moderate depressant actions on the EEG and the behavior in cats. They were qualitatively similar in their actions, although Rg 1, Re and Rb 2 were more potent than the rest. They also suppressed EEG arousal response induced by electrical stimulation of the mid brain in cats.


Pharmacological properties of n1-piperonyl-n4-3,7,11-trimethyl-2,6,10-dodecatrienyl-piperazine, a new non-anticholinergic gastric antisecretory agent.

The special and general pharmacology of N1-piperonyl-N4-3,7,11-trimethyl-2,6,10-dodecatrienylpiperazine (U-27) is reported. According to the results of the animal experiments the compound turned out to be a well tolerated gastric antisecretory drug devoid of anticholinergic activity. The compound was able to prevent hypersecretion induced by pylorus ligature in rat and guinea pig. Its duration of action was remarkable and no tolerance occurred after a repeated treatment. The compound displayed also an interesting activity on several experimental ulcers.


[Stability of amphetaminil. 2. In vivo studies].

The metabolism of radioactively labelled alpha-phenyl-alpha-N-(beta-phenyl-isopropyl)-aminoacetonitrile (amphetaminil, AN 1-R) (tritium-labelled in the amphetamine-part, 14C-labelled in the benzaldehyde-part of the molecule) in the rat was examined. In the body amphetaminil is cleft very quickly into the original compounds of its chemical synthesis (amphetamine, benzaldehyde and hydrocyanic acid) independent of the mode of application (i.p. or oral). Blood: In the interval from 5-90 min after application of amphetaminil only 1-2 percent of the total radioactivity are received from amphetaminil. The main part of the tritium- and 14C-radioactivity is distributed among amphetamine, p-OH-amphetamineglucuronide and hippuric acid. Brain: In the brain only a minimum amount of amphetaminil can be present, if any. The share of amphetamine of the total activity amounts to more than 90 percent. Adipose tissue: In the adipose tissue the ratio of the two isotopes of the substances extracted at pH 5 is almost equal to that of the administered amphetaminil. It seems that the substance is enriched there because of its pronounced lipophilia. After i.p. application the concentration is about 12 times higher than after oral application. Urine: In the urine there could be detected amphetamine, p-OH-amphetamineglucuronide and hippuric acid but no amphetaminil.


The contribution of serum enzymes and carcinoembryonic antigen to the early diagnosis of metastatic colorectal cancer.

The evolution of metastatic colorectal cancer in patients who have had surgical treatment for a primary lesion was studied in relation the progressive changes in the blood levels of carcinembryonic antigen (CEA), to gamma glutamyl transpeptidase (GGT) and routine liver function tests (LFTs). Involvement of the liver could ofter be reliably predicted many weeks in advance of clinical diagnosis while metastases to other sites were less likely to be detected early by this test. The association of the extent of disease with the patterns of biochemical changes is discussed with reference to several illustrative examples.


Extraction and preliminary characterization of a human bronchogenic carcinoma antigen.

Saline extracts of human bronchogenic tumours, soluble in 50% saturated ammonium sulphate and also fractions from Sephadex G-200 chromatography were used to raise antisera in rabbits. After absorbing the antisera with normal tissue extracts, direct Ouchterlony tests were performed against tumour (adenocarcinomata and squamous cell carcinomata) and normal extracts. A precipitin reaction was given with all 11 tumour extracts tested at a concentration of 5 mg/ml whereas all the 9 normal lung control extracts did not react at concentrations up to 100 mg/ml. The possibility that this reaction could be related to histocompatibility differences between individuals is ruled out by the fact that in two cases tumour and normal tissue were obtained from the same patient. These studies and also precipitin-inhibition experiments have confirmed the existence of antigen associated with bronchial carcinomata and have shown that, although the antigen or a cross-reacting antigen is present in normal lung tissue, the amounts are small in comparison with the amounts extracted from tumour. Antigenic activity was contained in a single absorbance peak when fractionated by Sephadex G-200 chromatography and its elution volume indicated a molecular weight of approximately 4-0 times 10(4)D. Further purification was achieved using isotachophoresis. Preliminary characterization of the antigen has shown it to be stable at pH 4-5, resistant to heating at 50 degrees C for 30 min, to migrate on immunoelectrophoresis with a cationic mobility at PH 8-5 and to be immunologically distinct from carcinoembryonic antigen.


Solar urticaria. A case with possible increase of skin mast cells.

A case of solar urticaria is described showing: (I) Action spectra for late erythema (MED), late swelling and wealing with one peak of sensitivity for erythema and wealing at 405 nm.  (2) No signs of porphyria. (3) Possibly increased skin mast cells. (4) Short-lived post-irradiation fibrin deposition. (5) Haemolysis. (6) Apparent suppression of urticaria with the antihistamine Incidal.


The bacteriological ecosystem of the skin of children in an African tropical envirovment (Tanzania).

Using a standardized method for sampling, the aerobic microbial flora of the skin of ninety-five children in Dar es Salaam, Tanzania, was studied qualitatively and quantitatively. The subjects comprised three groups; group A, thirty-seven normal infants (below the age of 1 year); group B, twenty-nine infants with various bacterial skin lesions; and group C, twenty-nine nursery school children between 4-5 and 7 years. The following observations were made: (I) The quantitative results indicated; (a) great variation between individuals, which was more marked among the females; (b) lower bacterial counts in infants than in older children; (c) counts were not affected by the presence of infection or state of cleanliness; (d) there were some differences between females and males and between different ethnic groups. (2) Qualitatively, the types of organisms isolated were similar to those reported elsewhere. There were differences in carrier rates of many organisms but these were most striking with Staphylococcus aureus and Staph. albus. It is concluded that there are great differences in carrier rates between persons in a tropical African environment and those studied elsewhere. These difference may be due to environmental, geographical or ethnic factors. Such observations call for more studies in this area to establish the normal flora.


Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi.

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...


Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.


Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes.

N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA. The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it. The alkylated RNA base was found to be adenine. The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.


ATPase and GTPase activities associated with the 5-S RNA-protein complex of Escherichia coli ribosomes.

Specific 5-S RNA-protein complexes were reconstituted from Escherichia coli 5-S RNA and 50-S ribosomal proteins. These complexes consist of 5-S RNA and two major proteins, namely E-L18 and E-L25. Analysis for enzymatic activities shows that ATP and GTP are hydrolyzed and that this hydrolysis is independent of elongation factors.


Alkali-induced reduction of the beta-cytochromes in purified complex III from beef heart mitochondria.

Approx. 40-50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.


Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.


The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This  inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.


Affinity chromatography of enzyme cofactors: the separation of NAD on immobilised dehydrogenase colums.

1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.


Partial purification and properties of thiamine pyrophosphokinase from pig brain.

Pig brain thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) was purified 260-fold over extracts of brain acetone powder. A direct, radiometric assay was used to follow the purification. By isoelectric focusing, the purified enzyme appeared to have an isoionic point of approx. pH 4.2, but these preparations were still not homogeneous by disc-gel electrophoresis nor by analytical ultracentrifugation. The purified enzyme has a broad pH optimum extending from pH 8.3 to 9.3 in 0.028 M phosphate/glycylglycine buffers. For optimal enzymatic activity, the ratio of magnesium to ATP must be fixed at 0.6, which suggests that for this ATP-pyrophosphoryl transfer reaction, the enzymatically preferred reactant may be Mg(ATP)6-/2. A preliminary study of the kinetics of the reaction reveals that the enzyme may function via a partial "ping-pong" mechanism; on this basis, dissociation constants for ATPt and for thiamine were evaluated. Pyrithiamine, butylthiamine, ethylthiamine, and oxythiamine appeared to be competitive inhibitors with respect to thiamine as the variable substrate, and their inhibitor dissociation constants were calculated. The relatively poor affinity of oxythiamine to the enzyme emphasizes the 4-amino group in the pyrimidine ring as one of the specificity requirements for thiamine pyrophosphokinase. Preliminary values for the apparent equilibrium coefficient of the thiamine pyrophosphokinase-catalyzed reaction, in terms of total species, has been approximated at several initial concentrations of reactants: e.g. K'eq,app = (see article) 9.66 - 10(-3) M; and [Th]initial - 1 - 10(-6) and 2 - 10(-6) M, respectively, where TDP, Th, t and eq represent thiamine diphosphate, thiamine, total concentration and equilibrium concentration, respectively.


Interaction of transition metal ions with ribonuclease A. II. The selective effects of Mn2+, Zn2+, Cd2+ and Hg2+ on the histidine magnetic resonance.

Zn2+, Cd2+ and Hg2+ inhibit ribonuclease but Mn2+ does not except at very high concentrations. By high resolution NMR one can detect in the pH range 5-8 the C-2 protons of histidines 105, 12, and 119. The inhibiting ions produce large shifts of the resonance of His-12 but not of His-105. On the other hand Mn2+ broadens the C-2 proton of His-105 much more than it does those of His-12 and 119. The selective shifts suggest that the mechanism of inhibition is binding at or near the active site of which His-12 and 119 are a part. The selective broadening is a consequence of binding of the Mn2+ to a site very far from the active site but closer to His-105.


Neurospora crassa invertase. A study of amino acids at the active center.

1. The effects on Neurospora crassa invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) of a variety of group specific reagnets and other potential inhibitors were determined during a search for an irreversible inhibitor of the enzyme. Aniline, pyridoxal, enzyme substrate and products did not inactivate invertase under reducing conditions. Bromoacetic acid, iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, hydroxylamine and 2-hydroxy-5-nitrobenzyl bromide were also ineffective. Iodine was the only reagent which irreversibly inhibited invertase. 2. Invertase was rapidly inactivated by low concentrations of iodine, indicating specific inhibition. However, the enzyme could not be protected from this inactivation by substrate. It was not reactivated by mercaptoethanol or cysteine. 3. Experiments on the uptake of radioactive iodine demonstrated that invertase is not iodinated under the conditions of iodine inactivation. 4. The sedimentation (S20,w) value of invertase was not altered by iodine inactivation. One-dimensional electrophoresis and finger-printing of tryptic digests revealed no differences between iodine treated and untreated invertase. There was no loss of carbohydrate from this glycoprotein during iodine inactivation. 5. Standard amino acid analyses of iodine-inactivated invertase showed some loss of tyrosine and a trace amount of methionine sulfone. Attempts to demonstrate oxidation of methionine to the sulfone, through modification of the procedure for preparation of samples for analysis, were unsuccessful. However, oxidation of half-cystine was indicated and further loss of tyrosine noted. A hypothesis is advanced that half-cystine is oxidized by iodine to a normally unstable oxidation state which is maintained and protected by its protein invironment and that loss of tyrosine may be an artifact caused by the presence of this residue during acid hydrolysis.


Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.


Purification of granulocyte neutral protease from human blood and rheumatoid synovial fluid.

The neutral protease activity of human synovial fluid cells, like that of peripheral blood leucocytes, is located in a granule fraction. It can be solubilised by various agents but only 1 M neutral salts do so without inactivation. Salt-solubilised neutral protease has been purified (300 X) from synovial fluid cells; like preparations obtained in the same way (600 X purified) from peripheral blood leucocytes, it has a broad pH profile of activity (pH 7--10.5) and in this, as well as in substrate specificity and sensitivity to activators and inhibitors, it behaves as a serine-histidine type protease similar to elastase (EC 3.4.21.11). The product showed two major components on polyacrylamide gel electrophoresis. Collagenase or chymotrypsin-like activity were not detected.


pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.


pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [14C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H+ greater than Cs+ greater than Rb+ greater than K greater than Na+ greater than Li+ greater than Mg2+, Ca2+. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondiffusible anions and (2) a selective permeability of the lysosomal membrane to cations.


The binding of fd gene-5 protein to single-stranded nucleic acid.

The gene-5 protein of the fd filamentous bacterial virus binds to single-stranded DNA over a pH range of 2-10.3. Binding to fd DNA is several hundred-fold stronger than to bacteriophage R17 RNA or to DNA tetranucleotides.


Quantitative separation of nucleotides on mercurated dextran.

Dextran gels of varying porosites (Sephadex G series) were chemically modified so as to contain covalently bound monofunctional mercury. Mercurated Sephadex of the porosity G-25 was then used to fractionate mixtures of mono- and dinucleotides into the constituent components. Separation is based on the affinity of the nitrogen binding sites of the purine and pyrimidine derivatives for organomercurial Hg+. Thus, a mixture composed of the four mononucleotides Cyd-5'-P, Ado-3'-P, Guo-2'(3')-P, dThd-5'-P and of the four dinucleotides Cyd-P-Cyd, Ado-P-Ado, Guo-P-Urd, and Urd-P-Urd could be separated into eight well-resolved fractions by using a combination Tris-bicarbonate/carbonate buffer system of increasing pH as an eluent. Complete separation was also achieved when a mixture of Ado 3:5'-P, Ado 5'-P, Ado-5'-PP, and Ado-5'-PPP was fractionated on mercurated Sephadex G-25. Again, Tris-bicarbonate/carbonate buffer served as an eluent. Lastly, fractionation can also be performed at a constant pH by offering other suitable ligands, for instance Cl-, that will compete with nucleotides for monofunctional Hg+. The fractionation behavior of mercurated Sephadex G-25 can be fully understood on the basis of the complexing properties of monofunctional Hg+. This has been shown by calculating the net retention volume ratios of several nucleotides with the help of the known interaction parameters of corresponding nucleosides with CH3 HgOH and by comparing the predicted ratios with the experimentally measured ones. Finally, the acid-base properties of mercurated Sephadex G-25 as well as its affinity for chloride and iodide ions have been determined. The data agree quite well with those known for CH3 HgOH.


Cardiac output measurement with a simplified thermodilution technique. Comparison with the Fick method.

A simplified thermodilution technique for cardiac output measurement is compared with the Fick method in dogs, over a wide range of cardiac output values. The reproducibility of the method is satisfactory (differences less than 10 per cent for 95 per cent of the measurements). The overall correlation factor (r = 0.97) is highly significant for cardiac outputs ranging from 750 to 7,500 ml.mn(-1). The influence of hematocrit (when lower than 30%) and of the injection site of indicator are discussed. Present experiments confirm that the use of pre-set factors allow for the thermal loss inside the catheter does not obviate the accuracy of thermodilution method for cardiac output measurements. The slope of the regression line is very close to 1. Lack of fit to linearity is tested and is found not significant throughout the explored range. This technique should therefore have extensive clinical applications.


Nonlinear regression methods in design of experiments and mathematical modelling. Applications to the analysis of the steady-state kinetics of glutathione reductase.

A branching reaction pathway involving a ping pong and a sequential loop has been proposed for glutathione reductase (Biochem. Biophys. Res. Commun. 53 (1973) 1151). In the present investigation nonlinear regression methods have been applied in the fitting of rate equations to experimental data to test the validity of the model proposed and to discriminate between alternative mathematical models (cf. FEBS Lett. 26 (1972) 252). In the best rate law, some of the parameters were numerically redundant. Therefore, a feature-wise analysis of the rate equation was carried out by varying one substrate concentration at a time. The overall strategy used was a cyclic procedure involving: experimentation - analysis of data - modelling - design of experiments - new experimentation etc. Consideration was given to the experimental error structure and to the importance of weighting in the regression analysis. In the design of experiments for discrimination between rival models, a previously defined discrimination function was used. The results of the analysis support the branching reaction scheme proposed for glutathione reductase.


Energetics of abiogenic chemical systems.

After recalling the energy consumption necessary to produce the main categories of biochemicals from the equilibrium state of an hydrogenated atmosphere, the primary processes by which energy can be absorbed in a mixture of methane and ammonia in the presence of aqueous solutions are defined. From the very first excitations, unsaturated products are formed. In fact, this formation of atmospheric precursors is the primordial state of a photochemically induced redox dismutation. The evolution of solutions obtained from the dissolution of these atmospheric precursors in aqueous media is described from experimental data and analysed on energetic grounds. The relaxation of energy accumulated in such solutions involves non enzymic archetypes of the main categories of metabolic processes; in particular some unsaturated atmospheric precursors must be looked upon as primordial representatives of biochemical dehydrating agents. Absorption of light energy in the solutions obtained from the evolution of precursors happens near the visible range and should govern their further evolution. In fact, biochemicals which were previously detected as products of model experiments are not present in these solutions. They were probably obtained during analytical procedures from products of the evolution of atmospheric precursors which are unstable against decreases of pH. Cyclic autocatalytic effects must be involved in the further evolution of the models of the first aqueous solutions. Their possible role in the appearance of optical dissymmetry is emphasized on theoretical grounds.


Endogenous oscillations in pathways of energy transduction as related to circadian rhythmicity and photoperiodic control.

Evidence is presented for endogenous rhythmicity in energy transducing sequences of cellular metabolism which result in a circadian rhythm in adenylate "energy charge" and redox state (NADPH/NADP). From phase dependent photocontrol of enzymatic activity and pyridine nucleotide pool-size levels it is concluded that light - via photoreceptor(s) of photoperiodic control - modulates energy flow under conditions where overall energy transduction displays a circadian rhythm. The results are discussed in relation to temporal organization of development in general.


Influence on clinical practice of routine intra-partum fetal monitoring.

An attempt has been made to monitor by continuous fetal heart rate according all women admitted in labour. Altogether 85% of the 1070 patients delivered at one hospital were monitored in 1973 and 92% in 1974. Perinatal mortality fell significantly from levels in preceding years to 15-8 and 11-7 per 1000 births, respectively, in 1973 and 1974. The fall was primarily due to the elimination of intra-partum stillbirths and a significant reduction in neonatal mortality. The incidence of caesarean sections also fell from 9-7% in 1973 to 5-8% in 1974. All patients should be monitored because it is impossible to predict reliably intra-partum fetal distress from maternal "high-risk" factors present before the onset of labour.


Diagnosis of dyspepsia from data collected by a physician's assistant.

This paper presents a study of the diagnosis of "dyspepsia" in 154 patients based on data collected at their initial outpatient attendance via an interview with a non-medically qualified physician's assistant. The reactions of patients to this type of interview were favourable, and the data recorded were as reliable as those recorded by clinicians. We conclude (1) that the data recorded by the physician's assistant are valuable diagnostically; (2) where these cannot be collected by a qualified physician, this task may be delegated to a non-medically qualified person; but (3) this interview should augment and not replace the traditional clinical interview.


[Hemoglobin oxygen transport during experimental acute hypercapnia (author's transl)].

The effects on hemoglobin oxygen transport of acute respiratory acidosis have been studied in dogs inhaling a gaseous mixture with 12% CO2 (O2 21%) for two to five hours. In a first series of experiments, it was shown that the shape of the oxyhemoglobin dissociation curve (ODC) was not modified by severe acidosis (pH congruent to 7) lasting for two and a half hours. The Hill number (N equals 2.6) did not change significantly. The aim of the second experimental series was to stuey the Bohr effect and the hemoglobin oxygen affinity (P50). The control value for the respiratory Bohr coefficient (B) was --0.54; neither after two hours (--0.52), nor after five hours of hypercapnia (--0.55) was it significantly modified. The P50 expressed at arterial pH was much increased in acidosis (congruent to 45 torr); when expressed at standard p/ 7.4, it was slightly but significantly decreased (congruent to 1 torr) at the fifth hour. At the same time there was a decrease (p smaller than 0.05) in the erythrocyte 2,3-DPG approaching 15 p. cent; on the other hand the ATP concentration did not change significantly. No significant individual correlation was found between P50(7.4), 2,3-DPG and mean hemoglobin corpuscular concentration. These results suggest that during severe respiratory acidosis neither a change in the shape of ODC, nor a change in Bohr effect do affect the hemoglobin oxygen transport. The main characteristic remains the decrease in oxygen affinity of hemoglobin, due to the erythrocyte [H+] increase induced by hypercapnia ; this phenomenon is observed as long as the 2,3-DPG decrease stays moderate.


Stereospecific binding of D-lysergic acid diethylamide (LSD) to brain membranes: relationship to serotonin receptors.

D-[3H]LSD binds saturably, reversibly, and with a high affinity (KD = 10 nM) to rat brain membranes. The association and dissociation rates of binding are temperature dependent and fastest at 37 degrees C. Binding is enriched in crude microsomal (P3) membranes. D-[3H]LSD binding is stereospecific as L-LSD, the psychotropically inactive enatiomer, is 1000 times weaker than D-LSD as a displacing agent. The potencies of other LSD analogues parallel their psychotropic activity with the exception of 2-bromo-LSD (psychotropically inactive) which is as potent as D-LSD in displacing bound D-[3H]LSD. Serotonin is the only putative neurotransmitter with affinity (ED50 = 3 muM) for the LSD binding site, and psychotropically active alkylindoleamines are also potent displacing agents. Destruction of presynaptic serotonin neuronal elements by lesioning the midbrain raphe nuclei does not chang the affinity or maximum number of detectable in vitro D-[3H]LSD bindind sites. The regional distribution in monkey brain of D-[3H]LSD binding and high affinity [3h]serotonin uptake, a marker for pre-synaptic serotonin nerve terminal density, shows some correlation. The most notable exceptions are cerebral cortical areas which are highest in D-[3H]LSD binding and only intermediate in [3h]serotonin uptake. Our evidence suggests that D-[3H]LSD binds to post-synaptic serotonin receptors.


Studies of suspected neurotransmitters in the vestibuloocular pathways.

Isolated fresh cat trochlear and oculomotor nuclei, which contain the axon terminals of inhibitory neurons whose cell bodies are in the superior vestibular nucleus (SVN), actively synthesize and store [3H]GABA, [14C]acetylcholine, [3H]dopamine and [3H]tyramine from labeled precursors of these compounds. Twelve to 14 days following lesions of the ipsilateral superior vestibular nucleus or its efferent pathway to the oculomotor and trochlear nuclei, at a time when there is extensive degeneration of superior vestibular nucleus axon terminals in these nuclei, the synthesis and storage of GABA in the ipsilateral trochlear nucleus is markedly reduced compared to that in the contralateral trochlear nucleus; the synthesis of acetylcholine, dopamine and tyramine is not measurably affected. The oculomotor nuclei, which unlike the trochlear nuclei receive a heavy bilateral projection from the SVN, show no asymmetric decrease after SVN lesions in their ability to synthesize any of the compounds tested. The data support the identity of GABA as an inhibitory transmitter in the superior vestibular nucleus-trochlear nucleus pathway.


Precipitation of calcium phosphates from electrolyte solutions.  III. Radiometric studies of the kinetics of precipitation and aging of calcium phosphates.

Precipitation and precipitate transformation in the system sodium phosphate (pre-adjusted to pH 7.4)--calcium chloride (25 degrees) was studied by means of radiometric analysis using 45Ca and 32P as tracers.  Changes in the pH and the total concentrations of calcium and phosphate were followed during solid phase formation and the data were used to calculate composition changes of the precipitates and their supernatants.  In all investigated systems two-step precipitation was observed, the precursor being more basic than the secondary precipitate.  The composition of the latter was mostly within the range of the composition of octacalcium phosphate.  The course of further chemical changes was dependent on the pH established during secondary precipitation.  The heterogeneous exchange of the radionuclides between the solid phase and their supernatant solutions was also followed as a function of time.  The results indicate that recrystallization through the mother liquid accompanied by composition changes is the dominant mechanism of equilibrium of the solid phases.


Studies on tyrosine hydroxylase in neuroblastoma in relation to urinary levels of catecholamine metabolites.

Tyrosine hydroxylase (TH) activity has been determined in 22 neuroblastoma tumors from 15 patients, in 1 pheochromocytoma, 20 adrenal glands, 10 other tumors and organs, and 4 specimens of sera. The enzyme activity was found only in the neural crest tumors and adrenal glands, but the levels were too low to be detected in the other tumors and in normal liver and kidney tissues. The average specific activity (mean +/- SE) of TH in 23 neural crest tumors was 0.559 +/- 0.101; in 20 adrenal glands was 0.418 +/- 0.124 nmol/mg protein/minute. In 13 patients with neuroblastoma and 1 patient with pheochromocytoma, both TH levels in the primary tumors and urinary excretion of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were studied. The urinary excretion of VMA by 10 of 13 neuroblastoma patients and by the patient with pheochromocytoma was significantly to markedly elevated above normal levels; excretion of HVA by 12 of 13 neuroblastoma patients was similarly elevated. These results indicate that tyrosine hydroxylase, an enzyme specifically located in the adrenal medulla and monoaminergic neurons, is also present in neuroblastoma, a malignant tumor of similar embryologic origin, and in pheochromocytoma. Not only can TH activity in these tumors be demonstrated in vitro, but the elevated urinary excretion of VMA and/or HVA by the majority of patients with these tumors also indicates TH activity of the tumors in vivo.


[The polyphosphate synthetase of Saccharomyces cerevisiae].

The polyphosphate-synthetase, isolated from a homogenate of phosphate starved cells, catalyses the synthesis of linear polyphosphates from orthophosphate. It is localized in the membrane fraction which deposits between 400 and 1000 X g; its optimal pH is 7.1; its KM toward orthophosphate is 4.0 X 10(-4) M; ATP stimulates the reaction. The enzyme synthezises especially polyphosphates with short chain length.


[Effect of hypercapnia on the acid base states of the blood in the crab Carcinus maenas (L.) (Crustacea-Decapoda)].

When the crab Carcinus maenas respires in hypercapnic water, a respiratory acidosis occurs, which is progressively compensated by a rise of the blood bicarbonate concentration. The CO2 partial pressure in the blood increases in proportion to the change in ambient CO2 partial pressure. Therefore, the regulation of the acid-base status depends mainly on non-respiratory adjustments of the blood bicarbonate concentrations.


[The effect of irradiation conditions on the radioinduced acidity of solid phase glucose].

Radioinduced acidity in solid state glucose is studied as a function of irradiation conditions (dose, dose rate, temperature, atmosphere composition), glucose characteristics (physical structure, water content) and post-irradiation treatments (presence of gas in dissolving water, storage).


Glycoside inotropy in the absence of an increase in potassium efflux in the rabbit heart.

The inotropic effect of 1.25 times 10(-6)M acetylstrophanthidin (ACS) and the influx and efflux of labeled potassium (42K+) were studied in the arterially perfused rabbit interventricular septum under control conditions and during respiratory acidosis. An increase in the CO2 content of the gas mixture with which the modified Ringer's solution  was equilibrated from 5 to 30% reduced the perfusate pH from 7.37 to 6.66. The increment in developed tension in the presence of ACS was 3.0 +/- 0.2 g (n equals 10) under control conditions, but it was greater, 7.1 +/- 0.9 g (N equals 9) during acidosis (P less than less than 0.001). The net K+ loss due to an increase in K+ efflux was 1.8 +/- 0.2 mmoles/kg wet weight in control experiments but only 0.1 +/- 0.1 mmoles/kg net weight under acidotic conitions (P less than less than 0.001); in seven of nine experiments in respiratory acidosis, no increase in K+ efflux occurred despite a marked positive inotropy. In three septums, K+ influx was reduced by ACS during respiratory acidosis. These results demonstrate that during acidosis ACS inhibits sodium-potassium adenosinetriphosphatase (Na+-K+ ATPase) and causes an inotropic effect but does not increase K+ efflux. K+ efflux cannot be linked to calcium (Ca2+) influx or regarded as the controlling factor of glycoside-induced inotropy. The results give further support to the proposal that digitalis-induced inotropy is secondary to an enhancement of a Na+-Ca2+ exchange system.


Noninvasive assessment of aorta-coronary saphenous vein bypass graft patency using directional Doppler.

Noninvasive monitoring of patency of aorta-coronary bypass grafts can be achieved with reasonable accuracy using the continuous wave directional Doppler and a pencil probe. The character of the graphic record and the auditory signal generated by the flow through these new vessels perfusing the myocardium are distinctive because of both the pattern of flow and also the fixed and relatively immobile position of these grafts in the anterior mediastinum. A total of 226 aorta-coronary bypass grafts were monitored in the first postoperative week: 82 to the right coronary artery (RCA), 90 to the left anterior descending (LAD), and 56 to the circumflex and lateral ventricular branches. Interpretation of patency in these latter vessels was consistently unreliable and was abandoned early in the study. This unreliability was probably due to their short superficial course and their juxtaposition to the aorta and the pulmonary artery. In contrast, flow through grafts to the RCA and the LAD could be established in approximately 90%. Of 82 grafts to the RCA, flow could not be detected in four (5%) and was doubtful in two (2%). In 90 grafts to the LAD, no flow was observed in four (4%) and was doubtful in five (6%). Comparing noninvasive Doppler results with angiography in 34 grafts, no false negatives were found, but there were two or 10% false positives. Flow characteristics in a functioning internal mammary implant were also found to be quite different from those in an intact opposite mammary artery. This form of monitoring requires some practice and experience but is quick, cheap and noninvasive. It offers an acceptable degree of accuracy in monitoring aorta-coronary bypass grafts to the RCA and the LAD when the conduits are in the anterior mediastinum.


Blood flow and vasoactive drug effects in internal mammary and venous bypass grafts.

Cardiac output and pressure-flow measurements were made in left internal mammary artery-to-left anterior descending coronary artery grafts and left subclavian artery-to-left circumflex coronary artery vein grafts after proximal coronary artery ligation. Similar measurements were also made in left internal mammary artery-to-left anterior descending coronary artery grafts and aortocoronary saphenous vein grafts in patients with coronary artery disease. Hemodynamic parameters and graft blood flows were measured during control baseline conditions and after pharmacological interventions using isoproterenol, phenylephrine, and epinephrine in random order. Blood flow through vein grafts and internal mammary artery grafts was similar under basal conditions and increased similarly with each of these vasoactive agents. Other hemodynamic parameters changed variably with the various pharmacological agents used. There was no close correlation between mammary artery or vein graft blood flow and arterial mean pressure and cardiac output over the wide range observed with the different drugs was associated with either an increase in perfusion pressure or a decrease in coronary vascular resistance. If the percentage increase in coronary blood flow above the basal control value in response to pharmacological stress is considered as a reflection of coronary vascular reserve, these studies suggest that coronary vascular reserve in patients with coronary artery disease is less than that of the normal dog heart.


The effect of vagus nerve stimulation upon vulnerability of the canine ventricle: role of sympathetic-parasympathetic interactions.

The effect of vagus nerve stimulation (VNS) upon ventricular vulnerability was studied in 30 mongrel dogs subjected to varying levels of adrenergic stimulation. Vulnerability was assessed both by determining the minimum current required to produce ventricular fibrillation (VF threshold) and by plotting VF threshold throughout the vulnerable period (VF zone). Chloralose-anesthetized animals were studied by means of sequential pulses applied to the apex of the right ventricular endocardium. Testing was carried out in closed-chest dogs, in open-chest dogs with and without left stellate ganglion stimulation (LSGS), and in open- and closed-chest dogs pretreated with propranolol. In the absence of adrenergic stimulation. VNS was without significant effect on either the VF threshold or the VF zone under closed- or open-chest conditions. During LSGS, however, VNS was associated with a 93 +/- 22% (mean +/- SE) increase in VF threshold (P less than 0.01) and constriction of the VF zone. Vagus nerve stimulation combined with LSGS raised VF threshold to the control value, but not beyond. After beta-adrenergic blockade with propranolol, VNS was without effect on VF threshold in either open- or closed-chest animals. It is concluded that augmented sympathetic tone is a precondition for a VNS-induced elevation in VF threshold. The vagal effect is indirect and is expressed by opposing the effects of heightened adrenergic tone on ventricular vulnerability.


Lack of significant effect of L(+)-glutamate on serum gamma-glutamyltransferase activity determined in the presence of glycylglycine.

No increase in gamma-glutamyltransferase activity determined in human sera at 25, 30, or 37 degrees C could be demonstrated when L(+)-glutamate, 10 to 5000 mumol/liter, was incorporated into reaction media containing glycylglycine, 40 mmol/liter.


Ultraviolet spectrophotometry of theophylline in plasma in the presence of barbiturates.

Ultraviolet spectrophotometry is widely used for the analysis of theophylline in blood, for purposes of monitoring therapy, and for pharmacokinetic studies. Phenobarbital, a component of common oral theophylline preparations, interferes with assays now in use. The modification of the method of Schack and Waxler [J. Pharmacol. Exp. Ther. 97, 283 (1949)] presented in this paper eliminates barbiturate interference. After solvent extraction and back extraction into NaOH, the pH of the alkaline solution is adjusted from pH 13 to 10 before spectrophotometric measurement. This shifts the barbiturate absorption maximum from 255 to 240 nm, permitting accurate analysis of theophylline, the spectrum of which is unaffected by the pH change.


A successful new primary health care provider in the newborn nursery.

The mothers' acceptance of the Child Health Associate ("CHA") as a qualified health professional and the documentation of CHA's clinical abilities to provide broad-based health care for normal infants attest to their capabilities to function as primary health care providers in a newborn nursery. In "low-risk" nursery settings, where other capable health professionals may not be available, the CHA can assume primary and direct responsibility for the care of the normal newborn infant.


Normal cardiac output during beta blockade with timolol in hypertensive patients.

Data published by this laboratory indicated that the beta adrenergic blocking drugs timolol and propranolol exerted equivalent beta blocking and antihypertensive actions in patients with mild essential hypertension, but that whereas cardiac output fell acutely with both drugs, it returned to normal after 5 wk of treatment with timolol, but remained depressed after propranolol. This preliminary observation needed further confirmation in a larger series of patients. In this study, 11 patients with initial diastolic blood pressures between 90 and 125 mm Hg were given timolol for 5 wk. Hemodynamic measurements were made before and at the end of treatment. Mean heart rate fell from 76.8 to 64.3 bpm (p less than 0.001), and blood pressure was reduced from 179.4/99 mm Hg to 167.4/93.3 mm Hg (less than 0.02). Cardiac  output averaged 6.29 L/min before timolol, and fell to 5.95 L/min (NS) after treatment. Stroke volume increased significantly, while total peripheral resistance was unchanged. These results confirm our earlier observations that timolol is an effective beta adrenergic blocking drug with antihypertensive action that does not reduce cardiac output significantly when given chronically. This drug merits further evaluation in other cardiovascular disorders since it may have advantages over other beta adrenergic blocking drugs.


Respiratory effects of lorazepam, pentobarbital, and pentazocine.

The respiratory effects of a new benzodiazepine, lorazepam, were compared to those of pentobarbital and pentazocine.  Pentobarbital, 50 and 150 mg, produced respiratory depression, as did pentazocine, 30 mg intramuscularly.  Lorazepam at 1.33 and 4 mg intramuscularly produced none.


Activity of doxycycline against respiratory pathogens.

149 patients were admitted from the Brussels area to the multi-centre Pan European trial of doxycycline in respiratory tract infections. Of these, 40 satisfied the strict criteria for detailed bacteriological study with positive culture of a known pathogen before treatment and bacteriological follow-up after treatment. The pathogens were beta-haemolytic streptococcus, group A (29 cases), pneumococcus (6 cases) and Haemophilus influenzae (6 cases); in one case, the pneumococcus and H. influenzae were present together. The results of doxycycline therapy were excellent, this included both its in vivo and in vitro activity; only one strain of pneumococcus proved resistant.


Doxycycline in respiratory tract infections. A review of a multi-centre trial.

A total of 256 patients were admitted in Germany into a Pan-European multi centre trial of doxycycline in the treatment of infections of the respiratory tract. The diagnoses showed a preponderance of lower respiratory tract infections compared with other European countries, due largely to the fact that the participating physicians were pulmonary or internal medicine specialists. Doxycycline was given in a dose of 200 mg initially followed by 100 mg daily for 5-10 days (only the occasional patient varied from this regimen). Side-effects were uncommon (2.5-3.5%) and affected only the gastrointestinal tract. Four patients had to change antibiotic therapy, not because of side-effects but because of possible bacterial resistance in these patients. The results were comparable with those from the rest of Europe: 88% of patients had a good or very good response. Nine patients were treated for longer than 14 days and only eight patients did not show improvement.


[Symptoms and clinical features of panarteritis nodosa (author's transl)].

Panarteritis nodosa was diagnosed in five patients, two of whom died shortly thereafter, while three are followed now up to four years. Clinical and biochemical findings will give the first diagnostic pointer to panarteritis nodosa, while the only proof can be by biopsy of the affected organ. Prognosis varies and must be assessed cautiously in the individual case. Type, extent and number of organs involved are of decisive role prognostically. An allergic-hyperergic reaction of the vessels walls is thought to be the cause. Corticosteroids and immunosuppressives may be used therapeutically. The neuromuscular form of panarteritis responds quite well to this form of medication, but prognosis depends on whether there is also visceral involvement.


An assay for erythropoietin in vitro at the milliunit level.

A method is described for the assay of erythropoietin using primary cultures of adult rat bone marrow cells. Either total labeled iron uptake by the cells or hematin synthesis from labeled iron may be used as the measure of erythropoietin action. The method is useful in the range 0.001 to 0.010 U, where the log response is linear with the log dose, and can be carried out in 2 working days. This method has the disadvantage of detecting asialoerythropoietin which has no activity in vivo.


Beta2-adrenergic stimulation of thyroid hormone secretion.

The influence on thyroid hormone secretion of the nonselective beta-adrenergic stimulant isoproterenol (IPNE), of a selective beta1-adrenergic stimulant, 1-isopropylamino-3-(2-thiazoloxy)-2-propanol (ITP), and of a selective beta2-adrenergic stimulant, terbutaline, was investigated in mice. A combination of light microscopy (colloid droplet formation) and bioassay (blood radioiodine--BRI--measurements) was used. IPNE and terbutaline induced formation of colloid droplets and increased BRI levels, whereas ITP was ineffective. The responses to IPNE and terbutaline were abolished or reduced by pretreatment with L-propranolol, but were not inhibited for pretreatment with D-propranolol or phentolamine. The results indicate that secretion of thyroid hormone can be induced through the mediation of beta2-adrenergic receptors.


Influence of the neuroleptanalgesic combination of etorphine and acepromazine on the horse: blood gases and acid-base balance.

Respiratory function and acid-base variables were studied in Welsh Mountain ponies before and at predetermined times after the intravenous injection of Immobilon and Revivon.A marked depression of respiratory rate was accompanied by large reductions in arterial blood oxygen tension and saturation and the development of a mild respiratory acidosis following the injection of Immobilon. It was concluded that at least three factors contributed to the hypoxic hypoxia produced by Immobilon; the posture of lateral recumbency, the decrease in respiratory rate and the laboured character of the respiration. Arterial oxygen and carbon dioxide tensions returned towards control levels soon after administering Revivon. Mixed venous oxygen tensions were little affected by either Immobilon or Revivon,  and mixed venous carbon dioxide tensions were increased to smaller degrees that those of arterial blood. Haemoglobin was increased initially by Immobilon, had returned to the control level by 30 min and fell below the control following the administration of Revivon.


Hematopoietic differentiative properties of murine spleen implanted in the omenta of irradiated and nonirradiated hosts.

Whole body irradiation of the recipients of syngeneic splenic implants into the omentum greatly enchances hematopoiesis and permits survial of and repopulation by stem cells of donor origin. Donor hematopioetic stem cells do not survive in spleen implants of the nonirradiated host; irradiated hosts were therfore used in the bulk of the experiments. Differentiation in the implants of splenic fragments is predominantly erythrocytic at 10 days and shifts to predominantly granulocytic differentiation at 21 days. Suspensions of spleen cells injected into the ometntum are predominantly granulocytopioetic at 10 days. The differntiation in fragments of spleen depleted of stem cells by irradtion, seeded with bone marrow cells and implanted into the omentum results in mixed erythocytic and granulocytic hematopoiesis, with granulocytic predominance. Lymphocytic cells appeared late in the implants of irrdiated recipients even at a time of prolific lymphocytopoiesis in the host's own spleens. The cause of the delay in the implants is not clear. The data are consisent with the concept that differntiation of hematopioetic stem cells is influenced by the stromal cells of the parent organ. The erythrocytic inductive capacity of the stromal cells may be lost by mechanical disruption or modified by irraidation or a prolonged period of implantation.


Evaluation of biologic activity of ferric chloride-treated endotoxin in mice.

Endotoxin (lipopolysaccharide, LPS) treated with ferric chloride was tested for its potential as a non-toxic agent for enhancement of non-specific host resistance. A 1 mg dose of untreated endotoxin, injected i.p. into mice, resulted in 100 per cent mortality, whereas the same amount of chemically-treated endotoxin resulted in less than 35 per cent lethality. The radio-protective potential of the treated endotoxin was similar to that of untreated endotoxin, as 70 per cent of each group of mice tested with either substance survived a dose of 850 rad x-ray. Irradiated mice, challenged 8 days after 850 rad x-irradiation, died when injected with 25 mug of either untreated or treated endotoxin. Antibiotic decontamination of the intestinal tract of host animals reduced the possibility of toxicity from endogenous endotoxin after challenge. This treatment resulted in 100 per cent survival from a 25 mug challenge at 8 days post-irradiation. The ferric chloride-treated proved to be a more effective B-lymphocyte mitogen. At a dose of 100 mug, treated endotoxin resulted in a 50 per cent greater mitogenic stimulation of B-lymphocytes as compared with that found after exposure to untreated endotoxin. Several lines of evidence support the contention that tolerance to untreated endotoxin was induced by repeated injections of either endotoxin preparation 1) 100 per cent of all endotoxin-tolerant mice survived a 1 mg challenge dose of untreated endotoxin, 2) there was a reduced mitotic response of splenic B-lymphocytes after re-exposure with untreated endotoxin as compared with that observed for cells derived from saline-treated mice, and 3) all antibiotic decontaminated mice engrafted with spleen cells from mice made tolerant to either endotoxin preparation survive graft-versus-host disease. In conclusion, based on survival data from normal mice, ferric chloride-treated endotoxin is safer to use than normal endotoxin. Also, treated endotoxin can elicit biologic responses similar in magnitude to those found after injection of mice with untreated endotoxin.


Gastric secretion during anaesthesia and its inhibition by metiamide and other drugs.

The histamine H2-receptor antagonist, metiamide, inhibits the acid gastric secretion produced by chloralose-urethane anaesthesia in dogs carrying gastric cannulae chronically. This secretion is also prevented by atropine and hexamethonium. "Spontaneous" gastric secretion of vagal origin in conscious dogs is also blocked by metiamide.


Uterine secretion during the sexual cycle in the rat and its capacity to disperse corona cells in vitro.

It was found that closure of the uterus disturbed the first 2 cycles after the operation; thereafter the normal cycle was resumed. The quantity of uterine fluid was increased at pro-oestrus and oestrus and reduced at met-oestrus and di-oestrus. Slight inverse changes in viscosity were observed. There was no significant difference in the pH. The corona-cell dispersing factor seems to be an oestrogen-dependent constituent of uterine secretion.


[A study on the determination method of cyclic AMP in urine by competitive protein binding assay (author's transl)].

The determination method of c-AMP in urine by competitive protein binding assay using the Boehringer Mannheim Laboratory kit was investigated and the results obtained were as follows. 1) Since the angle of the inclination of the standard curve was large, the present method could be used for the determination of c-AMP in concentrations of from 0 to 20p moles. 2) The optimal condition of the binding reaction was for 100 minutes of reaction time at pH 4.0 and 4 degrees C. 3) The specificities of binding protein to the other nucleotides were 0 to adenosine, 0.4 to AMP, 0.3 to ADP, 0.4 to ATP and 0.6 to c-GMP respectively when the specificity to c-AMP was chosen as 100. 4) The optimal volume of cold phosphate buffer solution needed for washing to separate binding c-AMP by filtration method using a millipore filter was 5 approximately 15 ml. 5) The precision of the present method by double determination was 0 arrroximately +/- 11.1% with average of +/-5.6% in c.v. 6) The recovery rate of the added c-AMP by the present method was 78.6 approximately 105.6% with average of 90.1%. 7) Correlation between the determination values of c-AMP of the same samples with the present method and radioimmunosasay (Schwarzman Laboratory Kit) was satisfactorily high with 0.890 in coefficient of correlation, and the determination values by the present method were significantly higher (p less than 0.05) than those by radioimmunoassay. 8) C-AMP in urine was stable for at least one month when the urine was kept frozen. It is conclusively considered from the above results that this competitive protein binding assay is a method to determine c-AMP in urine with excellent accuracy and sensitivity and that this method is useful for clinical test.


Effect of phenformin on gluconeogenesis from lactate and intracellular pH in the isolated perfused guinea pig liver.

Gluconeogenesis from lactate and hepatic cell pH (pHi) were measured in the isolated perfused livers of starved guinea pigs in the presence and absence of phenformin (phenethylbiguanide). The observed decrease in lactate consumption and glucose output in the presence of phenformin was associated with a fall in pHi. The fall in glucose output observed was considerably greater than accountable for by the decrease in lactate consumption. A possible mechanism for the pathogenesis of clinical lactic acidosis due to phenformin therapy is suggested.


Generation of free radicals of quinone group-containing anti-cancer chemicals in NADPH-microsome system as evidenced by initiation of sulfite oxidation.

The prerequisit of reduction for activation of Mitomycin-C and unstability of its reduced form suggested investigation of the possible formation of free radicals (semiquinone forms) of a series of quinone-containing anticancer chemicals in vitro. The ability of rat-liver microsomes to initiate sulfite oxidation in the presence of NADPH was markedly enhanced by the addition of these chemicals. This strongly suggests that these chemicals participated in the process in the form of reactive free radicals. The reaction was specific for NADPH. Carbazilquinone was unique among others in that NADH can replace NADPH and its higher ability to initiate sulfite oxidation. Microsomes from Ehrlich ascites and AH-109A hepatoma cells were also effective, though to a lesser extent than those from rat liver on a protein basis. Generation of free radicals, though their biological significance is not clear at present, may be deemed an inherent chemical property of these chemicals.


Comparison of gastric mucosal blood flow as determined by aminopyrine clearance and gamma-labeled microspheres.

Gastric mucosal blood flow was simultaneously determined by aminopyrine clearance and gamma-labeled microspheres (15 +/- 5 mu in diameter) in anesthetized dogs prepared with a chambered segment of stomach greater curvature. Paired flow measurements were made in 11 dogs (n = 28) secreting in response to intravenous histamine (1mug per kg per min), in 11 (n = 21) nonsecreting dogs given intravenous isoproterenol (0.5 or 1.0 mug per kg per min), and in 9 (n = 10) dogs given no drugs to stimulate secretion or blood flow (resting dogs). Eight additional injections were done in dogs receiving various combinations of isoproterenol and histamine. Isotonic HCl was maintained on the mucosal surface during all experiments. Regression analysis demonstrated a highly significant linear correlation between clearance and microsphere-measured flow in the histamine (P less than 0.001, r = 0.96) and isoproterenol (P less than 0.001, r = 0.78) experiments, with clearance averaging 83% of microsphere flow during histamine stimulation but only 25% during isoproterenol. The relationship between clearance and microsphere flow was not significantly different for the resting and isoproterenol experiments. Mucosal perfusion measured by microspheres was about 5 times the resting value for both histamine and isoproterenol-stimulated dogs. Perfusion calculated from aminopyrine clearance averaged 46, 38, and 90% of the microsphere value in the resting, isoproterenol, and histamine experiments, respectively. Pooled data from secreting dogs demonstrated a fairly constant ratio of microsphere-measured flow to clearance (1.25 +/- 0.06 mean +/- SEM), regardless of the secretory rate. Our results indicate that aminopyrine clearance reflects only a small fraction of mucosal blood flow in the nonsecreting stomach, even in the presence of exogenous acid.


Chemicals bathing the oxyntic gland area stimulate acid secretion in dog.

Release of gastrin is the only recognized mechanism by which chemicals in the stomach stimulate acid secretion. We report here that dietary components coming in contact only with oxyntic gland mucosa stimulate near maximal acid secretion through a local, H-sensitive mechanism that does not involve gastrin. In 4 dogs with gastric fistula and Heidenhain pouch (HP), 10% liver extract, 10% peptone, 0.4 M glycine, or Tris buffer, as control, was instilled into the HP in volumes of 40, 80, or 160 ml every 30 min. Instilled solutions were adjusted to pH 8.0 and HP acid secretion was measured by titrating a sample of the fluid recovered from the HP back to pH 8.0 with 0.2 M NaOH. Instillation of liver extract into the HP stimulated acid secretion from the HP but caused no change in serum gastrin and no change in acid secretion from the gastric fistula. The maximal response to liver extract occurred with the largest volume instilled and was 80% of the maximal response to histamine and 188% of the maximal response to pentagastrin. Expressed as per cent of maximal response to histamine, the maximal response to Tris buffer was 8%, to peptone 44%, and to glycine 14%. Intact bovine serum albumin gave no response, but after digestion by pepsin it stimulated acid secretion moderately. At pH 2.0, liver extract caused no stimulation of acid secretion. The pH threshold was about 2.5, and at pH 4.5 acid secretion was 55% of the response at pH 8.0. The response to liver extract at pH 8.0 was only minimally decreased by topical lidocaine or by intravenous atropine or metiamide. Since atropine and metiamide almost totally abolish the acid response to food in the main stomach, but do not inhibit secretion of acid evoked by instilling liver extract into the HP, there is reason to doubt whether this new mechanism operates under physiological conditions.


Distribution of Cryptococcus neoformans in a pigeon habitat.

Samples of pigeon droppings were taken from 7 sites in a church tower contaminated with C. neoformans, and the distribution patterns of the fungus were studied. From various sites, 0 to 3 x 10(5) viable C. neoformans particles were recovered per one gram of dry excreta. The factors causing the different density of C. neoformans population in the habitat were: uric acid share of the total nitrogen, creatinine content, sunlight and probably pH. Chemical composition of the substrate is the primary factor in the distribution of C. neoformans in droppings.


[Pharmacological action of [ethyl p(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY)].

General pharmacological effects of [Ethyl p-(6-guanidinohexanoyloxy)benzoate] methanesulfonate (FOY), a new antiproteolytic agent, were studied and the following results were obtained. Acute administration of large doses of FOY in conscious dogs and rabbits caused a decrease in spontaneous motility, ataxia, cyanosis, collapse, mydriasis, and respiratory paralysis. The agent had no effect on the central nervous system and exhibited hypotensive effects in dogs in doses of more than 1 mg/kg. Hypotensive responses were not inhibited by treatment with atropine or hexamethonium. FOY had no effects on ECG in the rabbit at doses of less than 30 mg/kg and at doses from 10(-6) to 10(-4)g/ml, distinctly reduced the amplitude of the spontaneous and rhythmic contractions of the isolated rabbit ileum, guinea-pig ileum and rat uterus preparation. The contractile response to nerve stimulation, noradrenaline and barium was suppressed in isolated guinea-pig vas deferens. FOY had no effects on the twitch response of gastrocnemius muscle to sciatic nerve stimulation in rats. The drug caused local irritant effects in rabbits and rats.


The activator of cerebroside sulphatase. Purification from human liver and identification as a protein.

1) A heat-stable activator of human sulphatase A (cerebroside sulphatase) was purified from human liver. It is required for the enzymatic degradation of cerebroside sulphates (sulphatides) in buffers (ionic strength greater than or equal 0.2) with osmolarity in the physiological range. 2) The purification steps involve extraction, acetone precipitation, heat treatment, isoelectric focusing and gel filtration. 3) Based on the definition of a specific activator unit, the purification of the final preparation was approximately 2000-fold over the acetone precipitation and several thousand-fold in the overall procedure. 4) The purified activator migrated as a single protein band when subjected to gel electrophoresis. Its effect was abolished after treatement with pronase E. The apparent molecular weight as determined by gel filtration was 21 500 +/- 1500; the isoelectric point was 4.3. 5) The activating effect of this protein factor and of taurodeoxycholate on cerebroside sulphatase activity was compared on a weight and molar basis.


Clinical and physiological assessment of chlorazepate, diazepam and placebo in anxious neurotics.

A 28-day double-blind assessment of chlorazepate dipotassium, diazepam and placebo was done on 30 outpatient neurotics with a primary symptom of anxiety. Acute, sub-acute and more chronic effects of the drug were assessed after 3 hours, 14 days and 28 days of drug administration. A battery of psychiatric ratings as well as physiological and psychophysiological assessments were done at each period. The psychometric assessments showed a trend for diazepam to be the most anxiolytic of the three drugs, followed by chlorazepate and then placebo. These measurements did not reach uniform statistically significant differences. Psychological measurements demonstrated the same trends, but some of these reached statistically significant levels. These parameters also indicate a slightly different mode of action of the two drugs at equimolecular doses. Diazepam would depress baseline and stimulation arousal, whereas chlorazepate would decrease baseline CNS arousal, but facilitate CNS response upon stimulation.


Amantadine in drug-induced extrapyramidal signs: a comparative study.

In our non-blind comparative study, amantadine was as effective as the standard medications, benztropine and ethopropazine, in controlling drug-induced extrapyramidal signs. However, statistically significant improvement was noted a week after all the three medications. In addition, extrapyramidal signs were not completely controlled in most patients even after weeks, even though substantial improvement was noted. This indicates the limitations of the currently available antiparkinsonian medications. Amantadine produced least side effects. Therefore, it may be particulary useful in patients who may not tolerate antiparkinsonian medication with anticholinergic properties. Our clinical finding that two patients with depression improved was rather interesting and needs further exploration. In summary, anamtadine is a valuable addition to our armamentarium of antiparkinsonian drugs. The drug compared favourably with other standard medications. The problem of dissipation of the therapeutic effects over time was not studied in our trial.


Spasmolytic activity of two synthetic anilide local anaesthetics.

Two basic anilides EA-7 and EA-8 were investigated for their antispasmodic activity against a variety of spasmogens on different tissues from different species of animals and comparison was made with lignocaine. EA-8 was found to be the most potent in this respect, followed by EA-7 and lignocaine. The antispasmodic potency does not correspond to their local anaesthetic potency. This suggests a direct depressant effect on tissues.


Study by PMR spectroscopy and gel chromatography of the unfolding of substituted kerateines in 8 M urea.

Various S-substituted derivatives of the reduced low sulphur and high proteins from wool have been prepared in which the substituted group is hydrogen, carboxymethyl, carboxethyl, methyl, carbamidomethyl, cyanoethyl and aminoethyl. The proton magnetic resonance (PMR) spectra and gel filtration chromatography of these proteins have been examined in 8 M urea solution as a function of pH in order to determine conditions under which the proteins occur as random coils in solution with no evidence for the occurrence of non-covalent interactions. The PMR method described in an earlier paper (1) provides an easier and much more sensitive method for the observation of non-covalent interactions in random coil proteins than does the measurement of elution volumes in gel chromatography. The results obtained by both methods are consistent and show that the widest range of pH for which unfolding occurs in 8 M urea is obtained with the S-carboxymethyl, S-carboxyethyl, S-methyl and S-carbamidomethyl derivatives.


Properties and immunochemical reactivities of carboxy-modified human serum albumin.

Seven carboxy modified and four amino modified derivatives of human serum albumin have been prepared and studied by optical rotatory dispersion measurements, gel filtration, immunoelectrophoresis, immunodiffusion and by use of an ammonium sulphate technique. It is found that carboxy groups are of major importance for the maintenance of the structure and function of human serum albumin, and that carboxy modification has a much more profound effect than amino modification has to the same relative extent.


The role of testicular conflicts in a countertransference reaction.

A case is presented in which a patient's presenting symptoms of impotence and a pervasive inhibition of productivity are dynamically related to a cryptorchild condition which was discovered and treated during childhood. The importance of the cryptorchid condition and its relationship to the patient's later psychopathology are examined. However, it is the therapist's reaction to the clinical material that is the paricular focus of his paper. The therapist obtained the history of cryptorchism in the fifth month of treatment, and then subsequently repressed this data. In addition, a persistent countertransference reaction developed that threatened to disrupt the treatment relationship. In reviewing the course of the therapy, it was possible to determine that the therapist's repression of the patient's cryptorchism and his subsequent countertransference response were directly related to his own repressed testicular conflicts. The implications regarding the existence of similar conflicts in other therapists are discussed.


Local graft versus host reactions within the anterior chamber of the eye: the formation of corneal endothelial pocks.

Introduction into the rabbit's anterior chamber of lymphoid cells sensitized to the histocompatibility antigens of the recipient leads to the development of an intraocular graft versus host reaction. This takes the form of a more-or-less severe uveitis, and the production of small focal areas of endothelial cell destruction on the posterior surface of the cornea. In the milder responses, these present as discrete pocks on the endothelial layer, which heal rapidly. In the more severe reactions, the foci of endothelial destruction may be so numerous as to overlap, resulting in complete immunologic destruction of the entire endothelium. The dose-response relationships in this experimental system are discussed, as are its uses as a model for the study of the mechanism of endothelial damage during the course of specific corneal graft rejection.


Prenatal metachromatic leukodystrophy.

In a family with a metachromatic leukodystrophy patient, two further pregnancies at risk were monitored by amnion cell culture. In one case, a normal baby was predicted and born. In the other case, a prenatal deficiency of arylsulfatase A was found. The diagnosis of metachromatic leukodystrophy was confirmed biochemically in various organs of the fetus by the deficiency of arylsulfatase A. The residual enzyme activity was shown to have an abnormal pH optimum and an increased heat stability. Ultrastructural studies revealed lipid storage in the myelinating nervous system and in the liver. For the interpretation of morphological results, it was indispensable to analyze an age-matched control fetus.


Comparative inhibitory activity of BL-S640 and two other cephalosporins.

In vitro antibacterial activity of BL-S640 was compared to that of cephalothin and cephalexin against Gram-negative and Gram-positive bacteria isolated from clinical specimens. BL-S640 demonstrated the best activity on nearly all microbial species studied, except for Haemophilus influenzae and Diplococcus pneumoniae against which cephalothin was slightly more active.


Semiautomated analysis of ergonovine maleate or methylergonovine maleate tablets and injections by colorimetric or fluorometric systems.

Two semiautomated analytical systems have been developed to determine ergonovine and methylergonovine maleate in single tablets and injections. The active ingredient is dissolved in a tartrate buffer. In the fluorometric method the stream is automatically diluted with tartrate buffer and excited with ultraviolet light; the resulting fluorescence is measured and recorded. In the colorimetric method, the stream is automatically made basic with sodium hydroxide and extracted with n-butanol. The extract is mixed with p-dimethylaminobenzaldehyde reagent. After reaction, the solution is mixed with sodium nitrite solution, and the developed color is measured at 550 nm. Recoveries of 100% were obtained from spiked placebos. Standard deviations for powdered tablet and injection samples ranged from 0.63 to 1.24%. Comparisons with the USP XVIII methods are presented.


Human erythrocyte glucose 6-phosphate dehydrogenase. Influence of coenzyme derivatives on thermostability and kinetic properties.

A number of derivatives of NADP(H) were tested with respect to their effectiveness in interacting with tetrameric glucose 6-phosphate dehydrogenase (G6PD) retaining only the fraction of "structural" coenzyme (4 moles NADP). Interaction was probed by two parameters: a) increased thermostability of G6PD activity, measured as the difference in the corresponding transition temperature (Tm) of samples containing and lacking the NADP derivatives, respectively; b) competitive inhibition toward NADP, expressed a Ki values. Protection afforded by the various effectors against thermal denaturation decreased in the following order: NADPH, NADP, PADP-ribose, adenosine 2',5'-P2. Other NADP derivatives, including 2',3' cyclic NADP, NMN, NMNH, nicotinamide, adenosine 2'-P, were uneffective in respect to this property. The kinetically measured affinity was the greatest for NADPH and decreased progressively for the following effectors: PADP-ribose, NADP, NMNH, PADP-glycolaldehyde, adenosine 2',5'-P2, PADP-ribitol, adenosine 2'-P. Nicotinamide and NMN were uneffective on NADP binding. These data show that the adenosine moiety of NADP is more critically involved than the nicotinamide portion in the interaction with human G6PD.


Soluble and membrane-bound aspartate-binding activities in Salmonella typhimurium.

The specificities of the soluble and membrane aspartate-binding activities were compared with each other and with the specificity of aspartate chemotaxis and were found to be distinct. The soluble aspartate-binding protein was purified to homogeneity and had a molecular weight of 30,000. The dissociation constant was 10(-6) M for aspartate, and the protein bound glutamate, cysteic acid, and 2-amino-3-phosphonopropionate. Aspartate transport was inhibited by cysteic acid.


Regulation of breakdown and synthesis of L-glutamate decarboxylase in Clostridium perfringens.

L-Glutamate decarboxylase (GAD) activity of Clostridium perfringens (ATCC 8009) cells grown in various culture conditions was investigated. Remarkable variations of GAD level occur during the growth cycle in thioglycollate broth. These changes are affected by the pH of the culture medium. Addition of alkali to the culture media results in decrease of cell GAD activity, whereas increase of enzyme level occurs only in cells growing in unbuffered media. The results indicate that the mechanism regulating the GAD levels is sensitive to the changes of pH (or buffering substances) rather than to the steady pH values. Neither repression by glucose nor induction by L-glutamate was observed. Moreover, high concentrations of the free amino acid substrate in the culture media considerably decrease cell GAD activity, owing to the buffering effect of the amino acid. The molecular mechanism supporting the variations of GAD activity during the growth cycle of the cells were investigated and tentatively related to the structural and functional properties of the pure enzyme. It is shown that the drop of GAD activity during the lag phase is due to protein breakdown. Evidence is presented suggesting a control of protein degradation by its quaternary structure. Data are also reported supporting de novo synthesis of GAD during the late logarithmic phase of cell growth. Finally, the possible role of GAD as part of the pH regulation system of C. perfringens cells is discussed in relation both to physiologic conditions of the bacterial cell and to the molecular mechanisms regulating the GAD activity in vivo.


Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes.

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.


p-Aminobenzoate-p-aminobenzoate.

p-Aminobenzoate (PABA) synthase from Bacillus subtilis is an aggregate composed of two nonidentical subunits and has the following properties. (i) In crude extracts this enzyme catalyzes the formation of PABA in the presence of chorismate and either glutamine (amidotransferase) or ammonia (aminase). The amidotransferase activity is about 5- to 10-fold higher than the aminase activity and is stable for at least 1 week when frozen at -70 C. (II) Although no divalent cation requirement could be demonstrated with crude extracts, 2 mM ethylene-diaminetetraacetic acid completely inhibits both activities. (iii) After ammonium sulfate fractionation both the aminase and amidotransferase activities require Mg2+ and guanosine in addition to the substrates indicated above for optimal activity. The guanosine requirement can be replaced by guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate but not by guanine, adenosine 5'-triphosphate, uridine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate, inorganic phosphate, and phosphoribosylpyrophosphate. Furthermore, at a pH above 7.4 or below 6.4 activity is rapidly lost a 4 C, or -60 C. (IV) The enzyme is composed of two non-identical subunits, designated subunit A and subunit X. Subunit A has an estimated molecular weight of 31,000, whereas subunit X has an estimated molecular weight of 19,000. Subunit A has aminase activity but no amidotransferase activity; a mutation at the pabA locus results in the loss of PABA synthase activity. Subunit X, which is also a component of the anthranilate synthase complex, has no PABA synthase activity itself but complexes with subunit A to give an AX aggregate that can use glutamine as a substrate. (v) The molecular weight of the AX complex has been estimated at 50,000, suggesting a 1:1 ratio of subunits. (vi) The enzyme is readily associated and dissociated.


Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae.

The localization of acid phosphatase (EC 3.1.3.2) in secreting protoplasts prepared from Saccharomyces cerevisiae is reported for the first time. Using a Gomori technique we were able to show acid phosphatase at those organelles in the protoplasts which are generally involved in the processes of biosynthesis and secretion of glycoproteins in eukaryotic cells.