A rapid and sensitive radioreceptor assay for benzodiazepine in plasma. We describe a rapid and sensitive radioreceptor assay for measuring benzodiazepines in plasma. This method is based on the competition between tritiated diazepam and pharmacologically active benzodiazepines present in plasma, for binding sites on rat brain synaptosomal membranes. No interference is obtained with drug-free plasma or plasma samples containing high concentrations of other commonly used drugs. High correlations (r = 0.98; P less than .001) were obtained between the "diazepam equivalents" measured in plasma with the radioreceptor assay and the levels of diazepam and nordiazepam obtained by gas-liquid chromatography. The radioreceptor assay is rapid, sensitive, specific, and requires no sophisticated equipment or methods. It should therefore prove useful in monitoring blood benzodiazepine levels for both therapeutic and toxicologic purposes.


Flurazepam effects on sleep EEG. Visual, computer, and cycle analysis. Analysis of sleep effects of flurazepam hydrochloride on four normal subjects confirmed that this drug substantially suppresses both REM and stage 4 sleep. Computer analysis disclosed that delta wave amplitude was greatly reduced by flurazepam. However, low density delta wave activity (ie, stage 2 sleep, which was increased in duration beyond the reduction in stage 4), permitted the number of delta waves and the time they occupied per night to remain at baseline levels. This finding suggests that sedative-hypnotics increase total sleep time by slowing the metabolic processes of sleep so that a longer sleep duration is required for the same biological effects. New observations on the induction times of REM and stage 4 effects are also presented. In general, the distortions in sleep EEG produced by flurazepam qualitatively resemble, but are quantitatively greater than, those produced by barbiturates in equivalent hypnotic doses.


A double-blind comparison of melperone and thiothixene in psychotic women using a new rating scale, the CPRS. Eighty-one women with psychosis of schizophrenic and paranoid type were assigned to a double-blind study comparing the clinical effects of melperone (100 mg X 3) and thiothixene (10 mg X 3). The antipsychotic effect was evaluated by clinical rating according to the CPRS and the NOSIE-30 scales before and after 2 and 4 weeks of drug treatment. A satisfactory interrater reliability was obtained for the CPRS. Significant correlation was also found between the CPRS and NOSIE ratings. Treatment with both drugs was associated with significant reductions in morbidity as estimated by several measures of therapeutic effect from the CPRS, by the NOSIE scale and by global ratings. There were no marked differences at any rating time point between the drugs in this regard. There were more extrapyramidal side effects in the thiothixene group than in the melperone-treated patients. The results encourage the use and further evaluation of melpherone in the treatment of psychotic patients.


Isolation of drugs from autopsy material by XAD-2 adsorption-elution technique. A routine procedure. The method of adsorption of drugs on Amberlite XAD-2 resin, followed by differential elution was under study during 1 1/2 year of routine use. The method allows a separation of acidic and basic drugs and--due to acid hydrolysis step before adsorption--assures better recovery of conjugated and protein-bound drugs. The amounts of various drugs, found in autopsy cases by the XAD-2 method were usually higher than those found by solvent extraction. The method applied requires 20 g of sample (biofluid or tissue) for general toxicological analysis.


Determination of monoureides in biological fluids by high-pressure-liquid-chromatography. A sensitive and specific method for the simultaneous determination of bromisoval, carbromal, and methaqualone is described. The drugs are adsorbed from serum onto charcoal at pH 11 and eluted from it with organic solvent. The eluate is separated by high-pressure liquid-chromatography on reverse phase (RP 18) column using acetonitrile: water (26 : 74 by volume) as mobile phase. The eluted drugs are detected by uv-absorption at 210 nm. The method is sensitive, specific, precise, and accurate.


Catalytic properties and stability of three common variants of placental alkaline phosphatase. Three placental alkaline phosphatases purified to homogeneity, i.e., the F, I, and S variants, were investigated for catalytic and stability properties. All three forms of the enzyme were found to have almost identical pH optima (10.7--10.8), similar sensitivity to the uncompetitive inhibitors L-phenylalanine (70%) and L-leucine (30%), and identical Km values against p-nitrophenylphosphate, beta-glycerophosphate, and alpha-naphthylphosphate. Significant differences among the three types were observed in thermal stability. The F variant was found to be most stable and the I variant most labile at 79 C. At 70 C all three forms were stable.


Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength. 1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.


A specific dimerization of rabbit beta-globin messenger ribonucleic acid. Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.


Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp. 1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).


The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide. The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.


Treatment of severe 2,4-D and mecoprop intoxication with alkaline diuresis. 1 Self-poisoning with a selective weedkiller containing 2,4-D and mecoprop in a 39 year old man resulted in prolonged deep coma, pyrexia, hyperventilation, hypoxaemia, myotonia, skeletal muscle damage and electrocardiographic changes consistent with cardiomyopathy. 2 The admission plasma concentrations of 2,4-D and mecoprop were 400 and 751 microgram/ml respectively. The patient remained gravely ill with no signs of improvement for 2 days with supportive therapy and there was no fall in the 2,4-D level. 3 Alkaline diuresis greatly increased the renal clearance of 2,4-D, and there was a rapid fall in plasma concentration (T1/2 3.7 h) with corresponding clinical improvement. The effect on the elimination of mecoprop was similar, but less dramatic. 4 Forced alkaline diuresis may improve the otherwise very poor prognosis in severe intoxication with 2,4-D and related weedkillers.


A pharmacokinetic study of doxapram in patients and volunteers. 1. Following intravenous bolus injections or brief infusions in healthy volunteers, plasma concentrations of doxapram declined in a multi-exponential fashion. The mean half-life from 4-12 h was 3.4 h (range 2.4-4.1h), the mean apparent volume of distribution was 1.5 1 kg-1 and the whole body clearance was 370 ml min-1. 2. Enteric-coated capsules of doxapram base were absorbed rapidly after an initial delay, and the systemic availability was about 60%. 3. Doxapram is extensively metabolized and less than 5% of an i.v. dose was excreted unchanged in the urine in 24 h. A metabolite (AHR 5955) was present in plasma in amounts comparable to the parent compound and had a similar half-life. 4. The disposition of doxapram appears to be similar in healthy volunteers and patients with respiratory failure. 5. The previously held belief that plasma concentrations fall rapidly when an infusion is stopped is only true following short duration infusions. The pharmacokinetic properties of doxapram are such that steady-state plasma concentrations will not be achieved for many hours with the recommended constant rate infusion régime.


The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis. The kinetics of the uptake and efflux of 3-O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate. The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 +/- 2 mM outside (Ko) and 18 +/- 2 mM inside (K1). The apparent maximum rate constants for the same system were 0.66 +/- 0.05 mmol/1 cell water per min for uptake (V+) and 0.53 +/- 0.04 mmol/l cell water per min for efflux (V-). For the active system K0 = 0.08 +/- 0.01, K1 greater than 40, V+ = 9.7 +/- 0.5 and V- = 1.1 +/- 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151--166). The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K0 And V+ in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.


Inactivation of the ribosomal protein S1 in polyuridylate binding by reductive methylation of the lysyl-ammonium groups. The ribosomal protein S1 was modified by reductive methylation of some of its lysyl ammonium groups (S1). With 6 out of 30 groups methylated the protein lost its capacity to form stable complexes with polyuridylate. Addition of excess polyuridylate inhibited the methylation of the lysyl groups. In equilibrium dialysis experiments it was shown that the binding constant between S1 and U15 was lowered 10-fold as compared to the native protein. The pH-dependence of the complex formation between S1 and U15 confirms a participation of the lysyl residues. When S1 depleted 30-S ribosomes were reconstituted with methylated S1 these ribosomes were inactive in the poly(U) stimulated Phe-tRNA binding. The data are discussed with respect to a grid-like interaction between the lysyl groups of the protein and the phosphodiester bonds of the polynucleotide as a molecular basis of protein nucleic acid interaction.


DNA-bound proteases. Partial characterization of proteolytic activities in commercial sources of DNA. The presence of variable amounts of protease in commercial sources of calf thymus DNA has been established by means of three independent methods; electrophoresis, substrate labelling and microfluorometry. The microfluorometric method, which is particularly useful for initial velocity measurements, has been used to characterize this proteolytic activity, either bound to DNA or solubilized by salt extraction. The proteolytic activity had a maximum at neutral pH and was not affected by NaCl concentration up to 0.5 M. Studies of specificity with natural substrates showed a higher V for protamine and lower Km for histones. The proteolytic activity was resolved by gel filtration chromatography in two major molecular weight species, differing in thermal stability and pH activity profile.


Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate. As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater  than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.


Delta14-sterol reductase in Saccharomyces cerevisiae. An in vitro assay for delta14-sterol reductase from yeast was developed, using ergosta-8,14-dien-3beta-ol as the substrate. The kinetics and localization of the enzyme were examined. The inhibition of the enzyme by the antimycotic agent, 15-azasterol, was verified.


Studies on the proton transport at system II in trypsin-treated spinach chloroplasts. The proton transport coupled with the DCMU-insensitive oxygen evolution mediated by K3[Fe(CN)6] in trypsin-treated chloroplasts (Renger, G. (1976) FEBS Lett. 69, 225--230) has been investigated with the aid of the pH indicator bromcresol purple. It was found that (1) the proton uptake from the outer aqueous phase observed in normal chloroplasts is completely suppressed by mild trypsin treatment; (2) a rather slow proton release into the external phase is detected which is insensitive to DCMU; (3) in the presence of DCMU, the extent of the proton release depends on the incubation time with trypsin in a similar manner as the average oxygen yield per flash. The results are interpreted by the assumption, that: (i) the reduced primary electron acceptor of System II, X 320-, does not become protonated, and (ii) the external acidification is caused by a passive efflux of protons, which are released by the watersplitting enzyme system Y into the inner phase of the thylakoids. The pK value of X 320- in trypsinated chloroplasts is estimated to be below 4.5. A possible pK shift caused by a modification of the proteinaceous barrier, which earlier (Renger, G. (1976) Biochim. Biophys. Acta 440, 287--300) was postulated to cover up the primary electron acceptor X 320, is discussed. Furthermore, the watersplitting enzyme system Y is inferred to be sensitive to deletereous attack from the outer aqueous phase mainly by secondary structural effects. Trypsination does not change the direction of the proton release in system Y.


Enzymic synthesis of a new type of fucose-containing glycolipid with fucosyltransferase of rat ascites hepatoma cell, AH 7974F. An alpha-fucosyltransferase activity has been demonstrated in rat ascites hepatoma AH 7974F cells catalyzing the transfer of L-fucose to asialo-GM1 prepared from bovine brain GM1 ganglioside to form a fucolipid in the presence of Triton X-100. The radioactive fucolipid was shown to be Fuc-(alpha1 leads to 2)-Gal-(beta1 leads to 3)-GalNAc-(beta1 leads to 4)-Gal-(beta1 leads to 4)-Glc-ceramide from the following results. The radioactive product coincided with authentic blood group H-active fucolipid from AH 7974F cell on thin-layer chromatography. The product formed a precipitation line not only with Ulex europeus lectin but also with eel anti-H serum on agarose gel plates. The terminal 14C-labeled fucose was released by Bacillus fulminans alpha(1 leads to 2)fucosidase as well as Charonia lampas alpha-fucosidase. The optimum pH value for the incorporation of L-fucose into asialo-GM1 was 5.8 in cacodylate/HCl buffer. The fucosyltransferase was highly specific for asialo-GM1.


Characterization of a soluble glycolipid galactosyltransferase which occurs in bovine milk. Bovine milk was found to contain, in soluble form, an enzyme which transfers galactose from UDPgalactose to glucosylceramide. This enzyme was partially purified by the same procedure used to isolate the galactosyltransferase of lactose synthetase. The partially purified enzyme required detergents for activity, had a pH optimum of 7.2--7.3 and required Mn2+. The apparent Km calculated for glucosylceramide was 1.33 . 10(-4) M. With glucosylceramide as acceptor the product of the reaction was identified as lactosylceramide by autoradiography on thin-layer chromatograms. Lactosylceramide was also an effective acceptor for the transferase reaction but neutral glycosphingolipids or gangliosides with terminal galactose of N-acetylgalactosamine residues were ineffective or poorly effective as acceptors. Addition of alpha-lactalbumin inhibited the transferase reaction.


Purification and characterisation of prostaglandin endoperoxide D-isomerase, a cytoplasmic, glutathione-requiring enzyme. Prostaglandin endoperoxide D-isomerase in rat spleen was purified until homogeneity. This cytoplasmic enzyme occurs in many organs of the rat and also in other species, and requires specifically glutathione for its action. The molecular weight is 30 000 and the isoelectric point pI 5.2.


Participation of superoxide, hydrogen peroxide and hydroxyl radicals in NADPH-cytochrome P-450 reductase-catalyzed peroxidation of methyl linolenate. 1. NADPH-cytochrome P-450 reductase-catalyzed peroxidation of methyl linolenate is inhibited by superoxide dismutase, catalase, ethanol, and mannitol, and is potentiated by H2O2. 2. H2O2 is shown to be generated in the incubation mixture in the presence of NADPH and NADPH-cytochrome P-450 reductase. If the system contains Fe-EDTA complex, H2O2 is not formed. In the presence of the enzyme and Fe-EDTA complex, added H2O2 is consumed. 3. In the presence of Fe-EDTA complex, NADPH-cytochrome P-450 reductase is shown to generate O-2 at a slow rate. These results suggest that H2O2 produced from O-2 is decomposed to form OH . by the action of Fe-EDTA complex in the lipid peroxidation system, and that OH . is a trigger of lipid peroxidation.


3-Hydroxy-3-methylglutaryl coenzyme A reductase from avian liver. Catalytic properties. The catalytic properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from avian liver have been investigated. Solubilized and highly purified reductase preparations were not cold labile, and enzymic activity remained unchanged following preincubation at 37 degrees C. The pH optimum was 6.8--7.0 and maximal catalytic activity was achieved with 2 mM dithiothreitol and 0.75 M KCl. The heat stability of the enzyme was studied and the addition of 0.75 M KCl, 0.8 mg/ml bovine serum albumin and 5 mM NADPH reduced the inactivation of the purified reductase associated with heat treatment at 65 degrees C. At 37 degrees C, 0.8 mg/ml bovine serum albumin enhanced the purified reductase activity by 100 (+/- 20)%. An improved assay was developed for the avian hydroxymethylglutaryl-CoA reductase and the specific activity of the purified enzyme increased from 1550 to 3300 nmol . min-1 . mg-1. The Km values of solubilized and purified reductase for D-hydroxymethylglutaryl-CoA were 1.05 micrometer and 1.62 micrometer, and for NADPH, 1 mM and 263 micrometer, respectively. The activities of the reductase preparations were non-competitively inhibited by coenzyme A, acyl-CoA esters, and hydroxymethylglutarate. MgATP also reduced avian reductase activity. These modulators may play a role in the cellular regulation of the reductase activity.


The thermal unfolding of ribonuclease A. A 13C NMR study. The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.


Investigation of transmitter-receptor interactions by analyzing postsynaptic membrane noise using stochastic kinetics. The stoichiometric and kinetic details of transmitter-receptor interaction (the number of conformations and the rate constants of conformation changes( in synaptic transmission have been investigated analyzing postsynaptic membrane noises by the aid of the fluctuation-dissipation theorem of stochastic chemical kinetics. The main assumptions are the following: (i) the transmitter-receptor interactions is modelled by a closed compartment system (a special complex chemical reaction) of unknown length,--(ii) the quantity of transmitter is maintained at a constant level,--(iii) the conductance is a linear function of the conformation quantity vector.--The main conclusion is: the conductance spectral density function is determined by three qualitatively different factors: (i) the length of the compartment system,--(ii) the precise form of the conductance-conformation quantity vector--(iii) the matrix of the reaction rate constants.


[Electrophoretic analysis of substrate specificity of wheat alcohol dehydrogenases]. Electrophoresis in polyacrylamide gel slabs has been used to study the isoform composition and substrate specificity of alcohol dehydrogenases in the embryo and young seedlings of the diploid wheat Triticum monococcum L., the tetraploid T. dicoccon (Schrank) Schuebl and the hexaploid T. spelta L. Three alcohol dehydrogenases of different substrate specificity and developmental pattern were distinguished: a) the NAD-dependent alcohol dehydrogenase, catalyzing the oxidation of different primary and secondary aliphatic and aromatic alcohols, as well as certain compounds with several hydroxyl groups (tris, triethanolamin) and revealing, after electrophoresis, one major band in the diploid wheat and three bands in both polyploid wheats; b) the NADP-dependent aromatic alcohol dehydrogenase (substrate--cinnamic alcohol), revealing, after electrophoresis, one major fast moving band in the diploid wheat and two bands in polyploid wheats; c) an aromatic alcohol dehydrogenase (2-3 bands after electrophoreis) with no specificity to the cofactors (NAD or NADP).


[Spectral parameters of the cytochrome c and hemoglobin interaction with methanol and aniline]. At 20 degrees C, in a phosphate buffer, pH 5,8--8,0, methanol and aniline interactions with hemoglobin and cytochrome c were studied using the difference spectrophotometry method. The difference absorption spectra are characterized by following values of lambdamax and lambdamin (nm): I--MeOH--hemoglobin (405 and 420), II-MeOH--cytochrome c (405--406 and 419--422), III--aniline--cytochrome c (421--410 and 401--396). The values of lambdamax and lambdamin for system III are shifted in the region of shorter wavelengths from 421 to 410 nm and from 401 to 396 nm, respectively within the pH range of 5,8--7,95. From difference spectra for systems I, II, III the dissociation constants of complexes obtained, Ks were calculated. Log Ks is linearly dependent on pH. System I is characterized by two values of Ks at all pH. The Ks values were calculated in general form from the dependences obtained. The nature of the complexes is discussed.


[Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]. The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.


[Metabolic activation of N-methyl-N-nitrosourea in normal and tumor cells]. N-methyl-N-nitrosourea is metabolised by mouse liver microsomes yielding highly reactive product(s) capable of alkylating cellular macromolecules. Based on cofactor requirement (NADPH, oxygen), inhibition (NaN3) and induction by phenobarbital, 3,4-benz(a)pyrene, and MNU, the reaction is cytochrome P-450-dependent. Analysis of the kinetics and dose dependence of alkylation in vivo and in tissue homogenates confirms the fact that MNU must undergo metabolic activation in vivo.


[Buffer capacity of polyproton substances]. Mathematical analysis of protolytic properties of a polyelectrolite with arbitrary set of stepwise dissotiation constants is carried out, based on disintegration of summary titration curves and buffer capacity for basic functions. It is demonstrated, that in some cases (for example, investigation of biomembranes and their components) simplified disintegration variants can be used. Buffer titration curve of biopolymers and biomembranes is a "buffer spectrum", the position of bands on the pH scale, their intensity and half-width being determined by the nature of the object (the quantity and quality of hydrophilic groups and the set of its conformation states). Sharp peaks of the buffer spectrum correspond to the conformation transition of the object, and their half-width on the pH scale determines the cooperative degree of each conformation transition. The mathematical analysis described is not specific to the formation of polyprotonic complex, and it can be used in investigation of the complex formation with any monodentant ligand. It is concluded that the method described can be used in the investigation of complex biomembranes and different processes, in which these membranes participate.


[Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]. Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.


[Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii]. Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.


[Interaction of aliphatid alcohols with cytochrome P-450 from rat liver microsomes]. The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed.


Cardiovascular reactivity of neonatal and adult sheep to autonomic stimuli during adrenergic depletion. The cardiovascular responses of the neonatal and adult sheep to autonomic agonists and antagonists were studied in the resting state and following adrenergic neuronal depletion with reserpine. Both the neonates and adults showed similar supersensitivity of the effector system of the peripheral circulation to alpha adrenergic stimulation with catecholamines. In addition, the neonates exhibited supersensitivity of the myocardial effector system to beta-adrenergic stimulation. Unusual finding was a marked supersensitivity of the heart and peripheral circulation to cholinergic receptor blockade with atropine, the mechanisms of which are as yet unclear.


Gastric response in low birth weight infants fed various formulas. Feeding techniques, delayed gastric emptying, volume overload, or reverse peristalsis may lead to regurgitation and aspiration in the premature infant. Noting these complications, various aspects of gastric function were studied in relation to the type of formula fed. 27 low birth weight infants (less than 2,000 g) were each fed one of three randomly assigned commercial formulas, which varied in osmolarity and MCT content. Gastric pH and emptying were monitored during the first 48 h of life. The fatty acid chain length of the triglyceride in the formula apparently did not influence either gastric pH or emptying. Infants fed formulas having a higher osmolar load (539 mosm/1) and containing protein hydrolysate showed greater gastric retention.


Perinatal development of the tyrosine oxidizing system. The enzymes of the tyrosine oxidase (TO) system of rat liver were measured during pre- and postnatal development. At all ages, the rate of tyrosine oxidation was limited by the activity of tyrosine aminotransferase (TAT). TAT of liver was undetectable from 4 days to 1 day before birth and was lower than p-hydroxyphenylpyruvate oxidase (pHPPO) and homogentisate oxidase (HO) postnatally. At birth, TAT, pHPPO, and HO were 2.9, 13, and 40% of adult values, respectively. Relative levels in fetal liver were HO greater than pHPPO greater than TAT. When enzyme activities were adjusted to compensate for dilution by hematopoietic cells, absolute activities increased, but other relationships remained the same. pHPPO rose at 15-17 days after birth and exceeded adult values at 31 days. HO was unchanged after birth. We conclude that each enzyme of TO follows a unique developmental path, with no concerted control of the entire TO system.


In vivo demonstration of renal carbonic anhydrase activity in the fetal lamb. The capacity of the fetal renal carbonic enzyme to facilitate the renal reabsorption of bicarbonate was studied in 8 long-term fetal lamb preparations (103-124 days of gestation). After acetazolamide administration the fetal arterial blood pH and PCO2 values decreased significantly (p less than 0.05). Significant increases in fetal urinary pH (p less than 0.001), and urinary excretion of bicarbonate (p less than 0.001), potassium (p less than 0.05) and total cation (Na+ + K+) were observed after carbonic anhydrase inhibition. It is suggested from these in vivo studies that renal carbonic anhydrase is present and active during fetal life and does not limit the capacity of the fetal kidney to reabsorb bicarbonate.


The quantitation of carbamino adduct formation of angiotensin II and bradykinin. The two equilibrium constants that define the extent of carbamino adduct formation with amines for all values of pH and PCO2 are determined for the alpha-amino groups of the peptide hormones angiotensin II(AII) and bradykinin (BK) by nuclear magnetic resonance techniques. From these constants the variation of carbamino adduct formation has been calculated over the pH range 6.60--8.00 with variable PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. The mole fraction, Z, of carbamino adduct form of AII or BK shows a maximum variation in going from metabolic alkalosis, Z congruent to 0.30, to metabolic acidosis, Z congruent to 0.02, with Z near 0.2 for normal acid-base conditions. Adduct formation to hormone may alter the biological effect of the hormone (a) by limiting proteolysis, particularly at the amino-terminal, (b) by altering hormone binding affinity to specific receptors, or (c) by converting the hormone to an antagonist which binds to receptor but does not activate subsequent metabolic events. The requirements for any of these mechanisms to operate are examined in terms of simple equilibrium considerations, and experimental evidence of inhibition of an aminopeptidase model system is presented. These results are consistent with the hypothesis that regulation of some physiological processes through formation of carbamino adduct of peptide hormones is possible.


Multimodal information processing in neurons. Advances in neuroscience research have led to substantial expansion and even revision of many concepts of neuronal structure and function. The essence of this progress is contained in the knowledge that there are multiple modes of information processing in neurons and even subneuronal elements such as the dendrite. The challenge and opportunity of the new findings lies in understanding how the various modes are integrated in complex brain function. Progress in this area should offer new insights into the pathophysiology of neurological disease.


The assessment of antichymotrypsin in cancer monitoring. A comparative study of serum antichymotrypsin, acid glycoprotein and antitrypsin levels has been made in various cancers. Antichymotrypsin (ACT) shows a non-specific elevation in metastatic cancer, and the response reflects tumour progression in a similar fashion to the other two acute phase reactant proteins. The main advantage of ACT as a marker appears to be its Normal distribution in health, and lack of phenotypic variation.


[Submicroscopic features of guanylate cyclase detection in mammalian epithelial and myocardial cells]. The electron cytochemical method of guanylate cyclase demonstrated a number of advantages making possible differentiation between the plasmalemmal, cytoplasmic, nucleoplasmic, and nucleolar localization of the enzyme. This method permitted to reveal that the enzyme possessed organ specificity of distribution in the cell and could be activated by a specific activator--sodium azide. The distribution of guanylate cyclase strictly correlated with the functional state of the cell.


An increased Bohr effect in sickle cell anemia. Recent findings that hemoglobin S gelation and sickling are pH-dependent and also influence oxygen affinity suggested that the red cells containing this hemoglobin variant might show an abnormal Bohr effect. We therefore studied the effects of pH variation on the in vitro oxygen affinity of whole blood from persons with sickle cell anemia (SS) and normal donors (at 37 degrees C and constant carbon dioxide tension of 40 mm Hg). The Bohr effect in SS blood was greatly increased only between blood pH 7.4 and 7.2 (cell pH 7.2 and 7.0, a shift that strongly affects gelation), with delta log p50/deltapH= - 0.92 to -0.99 (normal = -0.42 to -0.46). Thus a drop in SS blood pH below 7.4 in tissue capillaries yields twice the normal decrease in oxygen affinity and a large release of oxygen from red cells, whose risk of sickling is high. Even mild transient acidosis would seem hazardous for patients with sickling disorders.


Differentiation of the roles of histamine H1- and H2-receptors in the mediation of the effects of histamine in the isolated working heart of the guinea-pig. 1 Differentiation of the roles of histamine H1- and H2-receptors in the mediation of the effects of histamine on the isolated working heart of the guinea-pig was achieved through the use of histamine and selective histamine receptor agonists and antagonists. 2 Histamine over the dose range 10(-9) mol to 10(-6) mol produced dose-related increases in sinus rate, left intraventricular pressure (LVP)max, LVdP/dtmax, coronary flow, aortic flow, total cardiac output and external pressure-volume work. 3 Dimaprit, a selective histamine H2-receptor agonist, produced very similar responses to histamine. 4 2-Pyridylethylamine, a selective histamine H1-receptor agonist, had little effect on cardiac function unless large doses were administered. Such doses produced increases in all measured parameters. 5 Cimetidine, a selective histamine H2-receptor antagonist, antagonized the effects of histamine and dimaprit and some but not all effects of 2-pyridylethylamine. In the presence of cimetidine a decrease in all parameters with the exception of sinus rate was observed with both histamine and 2-pyridylethylamine. 6 The selective histamine H1-receptor antagonist, mepyramine, had little effect on responses to all three agonists. However, the depressant effects observed with histamine and 2-pyridylethylamine in the presence of cimetidine were antagonized by mepyramine. 7 The results indicate the important role of the histamine H2-receptor in the mediation of the gross cardiac effects of histamine and also indicate that histamine H1-receptors can mediate cardiac depression.


The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro. 1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content.


Lack of release of prostaglandins from isolated perfused lungs during pulmonary hypertension and oedema. 1 The effects on pulmonary prostaglandin synthesis of pulmonary hypertension and oedema have been studied. 2 Seventeen isolated lungs of rabbit, cat and guinea-pig were perfused with plasma, whole blood or Krebs Ringer solution in a recirculating system. 3 The venous effluent from the lungs superfused (10 ml/min) a series of smooth muscle tissues sensitive to prostaglandins and thromboxane A2; these were: rat stomach strip, rat colon, chick rectum and rabbit aorta. 4 Left atrial pressure was increased by between 10 and 30 mmHg for periods of 3 to 59 minutes. Gross alveolar oedema eventually developed in all experiments. 5 Neither pulmonary hypertension nor subsequent oedema caused release of prostaglandins into the venous effluent.


Prevention and reversal of isolation-induced systolic arterial hypertension in rats by treatment with beta-adrenoceptor antagonists. 1. Rats were made hypertensive by 5 days of continuous isolation in glass metabolic cages; in the text "hypertensive" means having a systolic blood pressure greater than 145 mmHg. 2. Daily intraperitoneal injections of either propranolol (5 mg/kg) or atenolol (5 mg/kg) reduced systolic blood pressure within 3 days and the systolic blood pressure remained low provided that the treatment was continued. 3. Treatment with metoprolol also lowered the systolic blood pressure of isolated rats but only when a larger dose (10 mg/kg) was given. 4. Systolic blood pressure returned to hypertensive levels following withdrawal of treatment after 15 days of isolation. However, following cessation of treatment after 27 days of isolation, the systolic blood pressure did not rise. 5. Rats given propranolol in the drinking water (intake equivalent to a daily dose of 5 mg/kg) before and during isolation did not develop hypertension. 6. The possibility that suppression of sympathetic function by the beta-adrenoceptor antagonists was responsible for these changes is discussed.


The sites of action of 5-hydroxytryptamine in nerve-muscle preparations from the guinea-pig small intestine and colon. 1 The sites of action of 5-hydroxytryptamine (5-HT) were examined in isolated segments of guinea-pig intestine. Mechanical records were taken from the longitudinal muscle of the ileum and proximal colon and from the circular muscle of the ileum and distal colon.2 In order to examine direct actions of 5-HT, nerve-mediated responses were blocked with tetrodotoxin (0.2 mug/ml). There was a gradient in the responsiveness of the longitudinal muscle of the ileum; in the proximal ileum it was usually unresponsive, whereas in the distal ileum about 30% of the amplitude of contraction was caused by a direct effect on the muscle. In the circular muscle from all parts of the ileum, direct effects on the muscle were weak or absent. In the distal colon, the circular muscle was almost always unresponsive to direct effects of 5-HT even when concentrations of 5-HT as great as 100 mug/ml were used. All direct actions of 5-HT on intestinal muscle were blocked by methysergide (1 mug/ml), which itself did not affect nerve-mediated responses.3 Excitatory cholinergic nerves and excitatory and inhibitory nerves which released unidentified substances were all stimulated by 5-HT. The contractions mediated through cholinergic nerves were blocked by hyoscine (0.6 mug/ml).4 Tachyphylaxis to the action of 5-HT occurred both for effects mediated through nerves and for direct effects on the muscle. Responses returned promptly after 5-HT was washed from the organ bath.5 While 5-HT blocked its own action on neural receptors, it did not antagonize the stimulation of nicotinic receptors on cholinergic neurones by 1-1 dimethyl-4-phenylpiperazinium iodide (DMPP). Moreover, pentolinium markedly reduced contractions caused by DMPP without significantly affecting responses to 5-HT. In contrast, (+)-tubocurarine, another nicotinic receptor antagonist, was effective in reducing contractions caused by 5-HT.6 Phenyldiguanide, which has been reported to antagonize the stimulant action of 5-HT on cholinergic neurones in the mouse small intestine, did not cause any significant reduction in the action of 5-HT on cholinergic neurones in the guinea-pig ileum unless a concentration of 1 mg/ml was used. However, contractions elicited by carbachol and DMPP were antagonized to a similar extent by phenyldiguanide at this concentration. Antagonism of the action of 5-HT at neural receptors by bromolysergic acid and by tryptamine was found but it was not specific, these drugs causing comparable decreases in responses to 5-HT, carbachol and DMPP.7 The present results, which show that 5-HT has little or no direct effect on the circular muscle of the ileum and colon, imply that, if 5-HT is a transmitter in enteric reflexes, it must be released from interneurones.


Treatment of irritable bowel syndrome with lorazepam, hyoscine butylbromide, and ispaghula husk. A double-blind controlled therapeutic trial of factorial design was used to study the therapeutic effects of lorazepam, hyoscine butylbromide, and ispaghula husk in 12 randomised blocks of eight patients with the irritable bowel syndrome (IBS). Each of the three agents caused a sustained symptomatic improvement in some of the patients, although only with ispaghula was the difference between the real and dummy preparation statistically significant. When the eight possible combinations of treatment were analysed none of the 12 patients who received only dummy preparations of the three agents had maintained any improvement over the three months of the trial. Seven patients improved among the 12 who received potent preparations of all three agents, and between four and six patients improved in the groups receiving one or two of the potent preparations. These therapeutic results, though far from perfect, show that the types of drug commonly used to treat IBS are of some value and may be additive in their effects. Similar combinations of other therapeutic agents may be more effective, but it will be possible to determine this only by carrying out factorial therapeutic trials.


Hair cell polarization in the gravity receptor systems of the statocysts of the cephalopods Sepia officinalis and Loligo vulgaris. The complete patterns of polarization of the sensory epithelia of the various gravity receptor systems of the decapods Sepia and Loligo have been described (Fig. 6). Each individual receptor cell (hair cell) bears up to 150 kinocilia, but is polarized unidirectionally by 3 morphological features: (I) by the orientation of the internal 9X2+2 tubuli structure of each kinocilium, (II) by the location of their basal feet. Each hair cell is additionally polarized (III), in that its kinociliary group is inclined toward the plane of the macula surface, forming an angle of 40-60 degrees with it (Figs. 1-3); the direction of polarization, as given by the ultrastructural features (I and II), is always opposite to this acute angle (Fig. 4). The results are discussed with reference to their physiological consequences.


Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain. Rotation-mediated aggregating cell cultures of mechanically dissociated fetal (15-16 days gestation) rat brains between 25 and 35 days in vitro were examined for their ability to synthesize neurotransmitters and putative neurotransmitters from radioactively labeled precursors added to the culture medium. Cultures derived from whole brain synthesized [3H]acetylcholine from [3H]choline, [3H]gamma-aminobutyric acid from L-[3H]glutamic acid, [3H]dopamine from L-[3H]tyrosine, [3H]dopamine and [3H]norepinephrine from L-[3H]dihydroxyphenylalanine, and [3H]serotonin from L-[3H]tryptophan. Veratridine increased and tetrodotoxin decreased the rate of [3H]-dopamine synthesized by aggregates derived from midbrain plus hindbrain. In chase experiments in which aggregates were incubated for 4 h with radioactively labeled precursors and then for 4 h with non-radioactively labeled precursors, addition of veratridine (50 micronM) during the second 4 h incubation significantly decreased the amounts of radioactively labeled acetylcholine, L-glutamic acid, dopamine and serotonin recovered from aggregates. Tetrodotoxin (5 micronM) present during the chase significantly increased the amounts of [3H]acetylcholine and [3H]dopamine recovered from the aggregates. In addition, reserpine (4 micronM) markedly depleted [3H]dopamine from aggregates in these experiments. These results indicate that these cultured cells synthesized neurotransmitters and in addition suggest that some of these compounds are stored by and released from electrically active cells within the aggregates.


Effects of fluoride on in vitro calcification of tendon matrix. Ca2+ and Pi uptake induced in vitro by a collagenous matrix derived from bovine tendon is inhibited by 1 X 10(-6) to 2 X 10(-5) M NaF and stimulated by 2 X 10(-5) to 2 X 10(-3) M NaF. Fluoride uptake occurs only over the latter concentration range. The uptake of Ca2+, Pi, and F-1 progresses toward a limiting extent at which the molar Ca/P and Ca/F values are 1.6 to 1.7 and 4.5 to 5.7, respectively. Although the matrix-bound mineral, previously formed in the absence of NaF, readily undergoes dissolution when exposed to a Ca2+- and P-free medium of pH less than 7.4, the bound mineral phase formed in the presence of NaF does not. We conclude that fluoroapatite is the primary matrix-bound mineral. The uptake of fluoride, Ca2+. amd Pi by both uncalcified and previously calcified matrices is inhibited by methylenediphosphonate and by phosphonoacetate as is calcification in the absence of NaF. Kinetic studies indicate that formation of a CaP complex precedes the uptake of F-1 and suggest that F-1 and OH-1 compete for interaction with that CaP complex during the calcification process. We concluded that fluoroapatite formation induced by the collagenous matrix occurs by a multistep pathway comparable to that proposed previously for hydroxyapatite formation.


Biological calcium phosphates and their role in the physiology of bone and dental tissues I. Composition and solubility of calcium phosphates. Variations in the composition of bone and tooth mineral are consistent with the model that the constituents are a mixed microcrystalline apatite (AP)-octocalcium phosphate (OCP) like phase and an amorphous or submicrocrystalline calcium phosphate (ACP) like phase whereby these phases can occur in different proportions. An appropriate model for a description of the variable composition and the solubility behavior of the apatite phase is given by the formula (formula: see text) in which the compositional parameters x, y, z, and u each account for one type of defect mechanism. Other point defects are formed as well by incorporation of minority amounts of ions such as Cl-, K+, and F-; a number of trace elements can substitute for Ca2+ ions under in vivo conditions. It is suggested that the incorporation of ions in or loss from the crystals in contact with aqueous solutions is reversible. Literature data are used to show the direction in which the solubility product of the apatite phase shifts by incorporation of the different physiologically relevant ions. A quantitative evaluation of the available literature data revealed that Na+ and CO3= incorporation is the main cause for shifts in the solubility product of biological apatites.


Midazolam compared with thiopentone as a hypnotic component in balanced anaesthesia: a randomized, double-blind study. Midazolam is a short-acting water soluble benzodiazepine derivative. It is a hypnotic used for intravenous anaesthesia induction. The present investigation was designed in a prospective double-blind fashion to compare midazolam with thiopentone as hypnotic components in balanced anaesthesia. The study included 50 healthy patients undergoing relatively short surgical procedures. The results revealed that thiopentone is faster in onset than midazolam for induction of anaesthesia, with less variation of dose response. However, maintenance of anaesthesia was superior with midazolam, requiring fewer supplemental anaesthetic drugs, having better patient acceptance and providing more amnesia. Postoperative complications were very low with both techniques. Midazolam was surprisingly similar to thiopentone in most parameters including emergence time from anaesthesia. Midazolam is a new drug with potential both for induction of anaesthesia and maintenance of balanced anaesthesia.


gamma-Glutamyl transpeptidase and malignant transformation of cultured liver cells. The relationship between gamma-glutamyl transpeptidase (GGT)and malignant cell transformation was analyzed in malignant and nonmalignant culture epithelial cell lines derived from rat livers and fibroblastic cell types derived from hamsters and mice. GGT activity was prominent (25 to 90% of cells) in 3 of 5 malignant epithelial liver cell lines. None of the 9 fibroblastic or 4 nonmalignant epithelial cell lines exhibited GGT activity. Our results suggest that by use of GGT activity we can detect in cultured liver cells a significant fraction of the spontaneously or chemically induced malignant cells. Thus, in conjunction with other markers, this marker may help in identifying tumorigenic cells in liver epithelial cultures.


[Changes in mammary tissue lipid metabolism in the goats during the first 2 months of lactation]. 5 French Alpine Goats were studied after normal or premature parturition. Mammary tissue acetyl-C oA carboxylase, glucose-6-phosphate dehydrogenase, malic enzyme and lipoprotein lipase (LPL) activities varied in parallel with milk fat secretion from the 3rd to the 9th week of lactation. Variations of mammary LPL activity and of long chain fatty acid secretion were positively highly correlated during that period. In goats with normal parturition, lipogenic activities seemed to reach a maximum level shortly after parturition. There was a positive correlation between mammary Ll activities and plasma non esterified fatty acid contents, possibly reflecting a relationship between adipose tissue mobilisation and mammary metabolism.


The dilating effect of histamine on pial arteries of cats and its mediation by H2 receptors. We studied the effect of histamine and H1 or H2 blockers on the diameter of pial arteries (39-227 micron) using microapplication into the perivascular space. Concentration-response curves for histamine showed dilations which started at 10(-7) M and were maximal at 10(-5) and 10(-4) M. The H2 blocker, cimetidine, induced no vascular reaction over the whole concentration range tested (10(-7) to 10(-3) M). The H1 blocker, mepyramine, was not vasoactive in the concentration range from 10(-7) to 5 X 10(-5) M and evoked dilations at higher concentrations. The concentration-response curve for histamine was only slightly displaced by 10(-5) M mepyramine but was significantly shifted to the right by 10(-5) M cimetidine. The dilating effect of histamine could be reduced in a stepwise manner by increasing concentrations of cimetidine. These findings are in accordance with a selective antagonism between histamine and cimetidine at the H2 receptors of smooth muscle cells of pial arteries. The insignificant role of H1 receptors in histamine-induced dilations is supported by the finding that a combination of H1 and H2 blockers resulted in the same reduction of histamine-induced dilation as did the application of the H2 blocker.


Influence of pH on the simple solubility test for myoglobinuria. We have investigated the effect of urine pH on the simple solubility test for myoglobinuria and found that the sensitivity of the test is very pH dependent. As much as a 10 fold increase in sensitivity can be obtained if the test is performed at pH 7.5 instead of pH 5.5. As such, we recommend that the urine pH should first be adjusted within the range 7.5--8.0 before performing the solubility test for myoglobinuria.


Cell-surface receptors in health and disease. Assessment of interaction of hormones and neurotransmitters with their cell-surface receptors can lead to an understanding of the molecular mechanisms that underlie diseases in man involving resistance and supersensitivity to these humoral agents and to pharmacological agents. They can also lead to the development of simple diagnostic procedures for such disorders, as well as the development of radioreceptor assays for humoral agents and drugs in blood and tissues, assays which combine sensitivity comparable to those involving radiommunoassay with a greater biological accuracy.


Automated enzymatic assay for plasma ammonia. An enzymatic method for determining plasma ammonia with the Du Pont Automatic Clinical Analyzer (aca) is described. The assay requires a sample volume of 500 muL for a kinetic ammonia measurement. The reaction is initiated with glutamate dehydrogenase and the rate of depletion of NADPH is monitored with two measurements, 17 s apart, at 340 nm. Reaction conditions have been optimized for maximum sensitivity through both one-factor-at-a-time and multiple variable response surface optimization techniques. Linearity to 1000 mumol of ammonia per liter of plasma has been achieved. No significant interferences were observed from anticoagulants or endogenous blood components, including pyruvate and oxalacetate. Use of the coenzyme NADPH (instead of NADH) in this aca procedure eliminates the lengthy pre-incubation otherwise required for endogenous dehydrogenase reactions.


Hydrolysis of glutathione by human liver gamma-glutamyltransferase. We studied the catalytic hydrolysis of glutathione by human liver gamma-glutamyltransferase [(gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2]. Glutamate production from glutathione was maximal at pH 7.4 (37 degrees C). Kinetically, the liver enzyme is similar to human kidney gamma-glutamyltransferase: their respective Km values with glutathione as substrate are similar (0.096 x 10(-3) mol/L and 0.097 x 10(-3) mol/L, respectively). S-Methylglutathione was hydrolyzed at a slightly higher rate than glutatione by liver gamma-glutamyltransferase. From these findings and other established properties of liver and kidney gamma-glutamyltransferase we propose that human liver is an important site of glutathione catabolism and that gamma-glutamyltransferase in liver catalyzes the first step of the catabolism of glutathione and glutathione conjugates in this organ.


Selective heat precipitability or reduced human hemoglobins in alkaline urea. Solutions of reduced hemoglobins from patients with either SS, SC, or CC hemoglobinopathies precipitated when heated for two minutes at 100 degrees C at pH 9.20 in the presence of 3.64 M urea, while hemolysates of normal adult AA and newborns (FA hemoglobin) remained clear. In patients with hemoglobin C trait (AC) having 30% or more C hemoglobin, precipitation also occurred. The conditions of pH and urea concentration affording precipitation of hemoglobin S approximated those observed for hemoglobin C.


A coupled enzymatic method to measure blood lactate by amperometric monitoring of the rate of oxygen depletion with a Clark oxygen electrode. A rapid, sensitive and reliable method to measure lactate in blood is described. The method is based on the enzymatic oxidation of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD+) and lactate dehydrogenase (LDH). The reaction product, NADH, is then oxidized by molecular oxygen, carried in the buffered reagent medium, in the presence of horeseradish peroxidase and other cofactors. The maximum rate of oxygen depletion, which is directly proportional to the amount of lactate ion present in the sample, is amperometrically monitored by a membrane oxygen electrode. No sample pretreatment is required in the present procedure other than dilution, and a comparison study between the described method and a spectrophotometric method shows good correlation.


Glucose-6-phosphate dehydrogenase deficiency in Sicily. Incidence, biochemical characteristics and clinical implications. This report deals with the incidence, type and clinical implications of G6PD deficiency in Sicily. Of 3347 male subjects examined, 56 were deficient in G6PD. They were distributed throughout the island. The G6PD levels in RBC were almost zero; in leukocytes, platelets and saliva they were found to be 26%, 18% and 16%, respectively, of controls. The Michaelis constant for NADP and G6PD was lower than for controls. Conversely, the utilization of the analogous Ga16P and 2dG6P was higher. The thermostability of the enzyme was lower and the pH optima (6.5 and 9.5) were different from the controls. An identical electrophoretic pattern was found both in normal and deficient subjects. This pattern is superimposable on that described as Mediterranean variant. The analysis among 270 subjects admitted to our Clinic with hemolysis due to G6PD deficiency demonstrated that the most frequent disease is favism, followed by neonatal jaundice, while hemolysis due to drugs is very rare. Ingestion of fresh fava beans was the most frequent cause of favism, but cases occurred after breast feeding and inhalation of pollen.


A study of the relationship between cardiac beta-adrenoceptor blockade and intrinsic sympathomimetic activity in rats depleted of catecholamines. 1. The intrinsic sympathomimetic activity of a range of beta-adrenoceptor antagonists and its relationship to beta-adrenoceptor blockade was studied in pentobarbitone-anaesthetized, vagotomized rats which had been depleted of catecholamines by pretreatment with syrosingopine. Dichlorisoprenaline, practolol, oxprenolol, pindolol and acebutolol, produced dose-dependent positive chronotropic responses in this preparation. 2. The relationship between the dose requirements for this intrinsic sympathomimetic activity and beta-adrenoceptor-blocking activity was not the same for all drugs: (i) dichlorisoprenaline and practolol had intrinsic activity at all beta-adrenoceptor-blocking doses; and (ii) oxprenolol, pindolol and acebutolol had predominantly beta-adrenoceptor blockade at the lower dose levels and agonist activity only became significant at high doses relative to those producing beta-adrenoceptor blockade. 3. The positive chronotropic response to both practolol and pindolol was observed in rats which had been pithed and was antagonized by propranolol (0.1-3.0 mg/kg, i.v.), indicating that beta-adrenoceptors were involved. 4. It was concluded that the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists was not a simple property as it was described by the relationship between the dose requirements for intrinsic sympathomimetic activity and for beta-adrenoceptor blockade as well as the degree of partial agonist activity.


The influence of the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists on haemodynamic effects in anaesthetized dogs. 1. The effects of propranolol, atenolol (ICI 66,082), practolol and pindolol on heart rate and maximal left ventricular dp/dt, atrioventricular conduction time, mean aortic flow and diastolic blood pressure during cardiac pacing were investigated over a wide dose range (0.025-4.0 mg/kg, i.v.) in dogs anaesthetized with pentobarbitone.2. Propranolol and atenolol produced similar reductions in haemodynamic parameters. Propranolol had no further effect in dogs pretreated with atenolol. 3. Practolol tended to cause smaller reductions in the haemodynamic parameters than either propranolol or atenolol. Subsequent administration of propranolol still had some depressant activity. 4. Pindolol produced a biphasic response, with depression of cardiac function at the low doses (0.025 and 0.1 mg/kg), but a reversal of effect as the dose was increased. 5. It is therefore concluded that, in anaesthetized dogs, the intrinsic activity of practolol and pindolol limits the fall in heart rate, cardiac conduction, aortic flow and maximal dp/dt observed with beta-adrenoceptor blockade. With pindolol, however, the influence of intrinsic activity is observed only in high doses related to beta-adrenoceptor blockade.


Cerebrospinal fluid lactate and pH in patients with acute severe head injury. Cerebral ventricular fluid (CSF) lactate and pH were measured repeatedly in 21 comatose patients with severe head injury during the first three weeks after trauma. In addition, regional cerebral 133Xe blood flow (rCBF) was measured two to four times in each patient at various time intervals, depending on the indications for carotid angiography, and the pressure in the cerebral ventricular system (IVP) was measured continuously. The series was divided into three groups: (1) patients with local cortical cerebral lesions, (2) patients with brain-stem symptoms, and (3) patients with both local cortical lesions and brain-stem symptoms. The results showed that a high CSF lactate level in patients with severe acute brain injury suggested severe and extensive brain lesions and predicted a poor outcome of the injury. In the individual patients, increasing CSF lactate foreboded clinical deterioration, whereas decreasing CSF lactate indicated recovery. The CSF pH was decreased in most patients, but very low pH levels (below 7.20) were seen only in three patients who all had a poor outcome. Correlation was not observed between CSF lactate and rCBF or between CSF pH and rCBF. It is concluded that repeated determination of CSF lactate and pH during the post-traumatic period may be a valuable tool in the assessment of the course and outcome of the brain injury.


An application of receptor models to the analysis of data on beta-adrenoceptor blockade. 1. The classical single receptor competitive occupancy model accurately describes the joint action of an agonist (isoprenaline) and a beta-adrenoceptor antagonist (propranolol) or some partial agonists (dichlorisoprenaline, practolol) on the positive chronotropic response in rats which have been depleted of catacholamines. 2. The mathematical form of the model suggests that the dissociation constants of classical competitive partial agonists may be assessed using dose ratios by exactly the same method as that currently used for agonist-antagonist interactions, provided that the log dose-response curves are first suitably normalized. 3. Close agreement between the theoretical mathematical models and the experimental data can be demonstrated by statistical fitting for certain beta-adrenoceptor antagonists (propranolol, dichlorisoprenaline, practolol). 4. The model fails to describe the behaviour of other beta-adrenoceptor antagonists (oxprenolol, pindolol). A possible extension of the model to include these drugs is proposed.


Acute effects of piretanide in normal subjects. Acute clearance studies were performed in normal subjects to assess the actions of the new diuretic, piretanide, on renal function. The drug increased both glomerular filtration rate and effective renal plasma flow in roughly proportionate amounts, so that filtration fraction did not change. In a dosage of 2 to 3 mg, it induced an increase in sodium excretion of almost 13% of filtered load, and there was an associated 2- to 3-fold increase in potassium excretion. The abstraction of solute-free water from the collecting duct was markedly reduced, but the drug induced no significant decline in the generation of free water. The rate of bicarbonate excretion, as well as that of titratable acid and ammonium, was increased approximately proportionately so that there was no increase in urinary pH or net hydrogen ion excretion. There was no phosphaturia, a unique finding, since all other drugs and maneuvers that cause a bicarbonate diuresis are also phosphaturic. Piretanide increased calcium excretion by approximately 19% of filtered load. The data suggest that the drug acts largely in the ascending limb of the loop of Henle and that it also affects the proximal tubule. Despite its sulfonamide structure, none of the drug's effects appear to be related to inhibition of carbonic anhydrase.


The effect of established beta-adrenoreceptor-blocking therapy on the release of cytosolic and lysosomal enzymes after acute myocardial infarction in man. 1. Serial venous blood samples were obtained from 45 patients with acute myocardial infarction. Ten of these patients were receiving beta-adreno-receptor-blocking drugs at the time of onset of chest pain and continued on these drugs during their stay in the coronary care unit. The activities of creatine kinase and its MB-isoenzyme (CK-MB) were assayed in the plasma. A lysosomal enzyme, beta-N-acetylglucosaminidase, was also assayed. 2. In the 35 untreated patients it was found that creatine kinase activity was maximal at a mean time of 21.3 +/- 1.3 h after the onset of chest pain, whereas in the patients receiving beta-adrenoreceptor-blocking drugs peak activity of the enzyme occurred at 24.4 +/- 0.7 h. 3. Peak CK-MB acitivity was also delayed from 18.1 +/- 1.6 h in the control group to 22.4 +/- 1.2 h in the treated patients. 4. The lysosomal enzyme showed a similar pattern of changes to that of CK-MB. Maximum activity in plasma occurred at 18.0 +/- 1.0 h after the onset of chest pain in the control group of patients. In the treated patients peak lysosomal enzyme activity was not found until 24.2 +/- 1.2 h. 5. These alterations in the time-course of plasma enzyme changes after acute myocardial infarction are consistent with the suggestion that beta-receptor antagonists may delay tissue damage during myocardial ischaemia.


Studies in the rat on the haemodynamic overshoot response to withdrawal of guanfacine or clonidine treatment. 1. Normal rats were injected intramuscularly twice daily for either 3 days or 3 weeks with guanfacine (0.1 or 1.0 mg/kg), clonidine (0.01 or 0.1 mg/kg) or 0.9% saline. All were anaesthetized at various times before or after the last injection, and their arterial pressures and heart rates were monitored via a carotid artery catheter. 2. Overshoots in systolic and diastolic pressure and heart rate, reaching peak values as soon as 16 h after the last injection, occurred in all rats withdrawn from guanfacine or clonidine treatment, but in no control rats. 3. Post-withdrawal blood pressures and heart rates of rats which had received the low dose of guanfacine or clonidine were as great as those of rats which had received the ten-fold greater dosage. Moreover, withdrawal responses were as great in rats which had been treated for only 3 days as in those treated for 3 weeks. 4. The dosages and duration of treatments used in these experiments thus did not influence the magnitude of the haemodynamic overshoots provoked by withdrawal of guanfacine or clonidine. However, all groups of rats from which guanfacine was withheld exhibited significantly greater peak overshoots in systolic and diastolic pressure than did those withdrawn from clonidine treatment.


Epidemiologic survey of accidental dentofacial injuries among U.S. Army personnel. This epidemiologic study of accidental dentofacial injuries to U.S. Army personnel was conducted to determine the frequency and distribution patterns of accidental dentofacial injuries to soldiers. Administratively, it was anticipated that this data would permit identification of high-risk groups and would suggest feasible preventive measures. This 9-month study was conducted on 16 Army posts with a combined population at risk of 210,500 soldiers; a standardized data collection form was completed by the dental corps officer treating the injury case and then was mailed to a central collection site for analysis. The data from this dentofacial injury study clearly reveal that differential risks exist for various military subpopulations. While the overall U.S. Army accidental dentofacial injury rate was 37.7 cases/10,000/year, this rate varied greatly for specific subgroups with high-risk factors including young males, lower enlisted ranks, recent recruits, and combat training posts. The primary specific causes of these injuries were fistfights (nearly 30%), sports (over 20%), and vehicles (about 15%).


Release of nickel from cooking utensils. The release of nickel to boiling water from new and used saucepans of different material was measured. No nickel was released from aluminium, teflon and enamel. Certain amounts of nickel were released from stainless steel, but only at acid pH.


alpha-Methyldopamine, a key intermediate in the metabolic disposition of 3,4-methylenedioxyamphetamine in vivo in dog and monkey. Metabolites of 3,4-methylenedioxyamphetamine in the urine of dogs and monkeys were separated by gas-liquid chromatography as their trifluoroacetyl and/or n-butyl ether derivatives and identified by comparison of the chromatographic and mass spectrometric behavior of these derivatives with those of synthetic compounds. The metabolites identified in dog and monkey urine were alpha-methyldopamine, 3-O-methyl-alpha-methyldopamine, and 3,4-dihydroxybenzyl methyl ketone. The monkey urine also contained 3,4-methylenedioxybenzyl methyl ketone and 3,4-methylenedioxybenzoic acid present as a glucuronide and/or sulfate conjugate, whereas the dog urine had 3-methoxy-4-hydroxybenzoic acid present as a conjugate other than glucuronide and sulfate. The phenolic metabolites in both species were present free and as glucuronide and/or sulfate conjugates.


Rabbit hepatic microsomal O-demethylation of chlorotrianisene. The in vitro hepatic O-demethylation of the nonsteroidal estrogen chlorotrianisene (CTA) has been studied. The rate of O-demethylation was maximal at 0.4 mM NADPH. Although NADH did not catalyze the reaction alone, it had a synergistic effect in the presence of equimolar amounts of NADPH. Carbon monoxide decreased the rate of O-demethylation. Nicotinamide was found to decrease the O-demethylation rate at a concentration of 40 mM, but had no apparent effect at concentrations of 20 mM or lower. Extracts from incubation mixtures contained one major (mono-O-demethylated) and a minor (bis-O-demethylated) metabolite. Extracts of mixtures containing soluble rather than microsomal enzymes or from mixtures in which microsomal protein had been denatured did not contain these metabolites.


Pharmacokinetics of sulfadimidine in normal and diarrheic calves. Pharmacokinetics of sulfadimidine after intravenous and oral administration of 50 mg per kg body weight were investigated in 2- to 22-day-old healthy and diarrheic calves. The data were processed in a digital computer. The values of absorption and elimination half-times after oral administration were 7.3 and 26 hr in healthy and 2.5 and 17.7 hr in diarrheic calves. It is evident from the results that the elimination of sulfadimidine in calves is very slow and characteristic of the species. In diarrheic calves the absorption half-time was significantly shortened and the same was found for the elimination half-time. The slow elimination must be taken into account when designing therapeutic schedules in calves. The effect of diarrhea on the pharmacokinetics of sulfadimidine indicate that more attention should be paid to changes of drug absorption, distribution, and elimination during diarrhea.


Evaluation of succinimidoethyl and pivaloyloxyethyl esters as progenitors of methyldopa in man, rhesus monkey, dog, and rat. The succinimidoethyl (Sm) and pivaloyloxyethyl (P) esters of methyldopa were evaluated as progenitors of the latter. Experiments in spontaneously hypertensive (SH) rats and humans demonstrated that a radioactive dose of progenitor was well absorbed. The metabolism of these progenitors appeared to be comparable in the SH rat; the urinary excretion of [3H]methyldopa was similar after oral administration of [3H]Sm or [3H]P. In humans the levels of [3H]methyldopa were higher in the urine following administration of [3H]P. Apparently Sm was more resistant than P to extrahepatic esterase action in man (and dog). In man the catechol nucleus of Sm was apparently conjugated prior to hydrolytic cleavage to release conjugated [3H]methyidopa. The progenitors possessed similar antihypertensive properties in the SH rat but preliminary results in humans suggested that Sm possessed less antihypertensive potency than P.


Biotransformation of prochiral 2-phenyl-1,3-di(4-pyridyl)-2-propanol to a chiral N-oxide metabolite. The prochiral compound, 2-phenyl-1,3-di(4-pyridyl)-2-propanol (PPP) labeled with 3H in the phenyl ring, was administered to rats, dogs, and a human subject. Paper chromatography of the urine indicated that a major metabolite common to all three species was excreted. This metabolite was isolated from the urine of chronically dosed dogs and was identified by mass, nuclear magnetic resonance (NMR), and infrared spectrometry as the N-oxide, 2-phenyl-1-(4-pyridyl)-3-(4-pyridyl-1-oxide)-2-propanol. In addition, polarimetry indicated that this metabolite was levorotatory. Examination of the enantiomeric purity of a crystallized sample of the metabolite by NMR spectroscopy of resolvable diastereomeric salts formed with lasalocid revealed the presence of only the levorotatory enantiomer. Accordingly, this metabolic N-oxide formation in the dog was at least stereoselective, and perhaps stereospecific. The N-oxidation of PPP was also demonstrated in vitro with 9000 g supernatant fraction of rat liver fortified with an NADPH generating system, and this reaction was inducible by phenobarbital, indicating that it is mediated by the cytochrome P-450 mixed-function oxidase system. This study, in addition to providing another example of the pyridyl N-oxidation pathway, illustrates the necessity of considering the stereochemical aspects of the metabolism of prochiral drugs.


The absorption, distribution, and excretion in mice of the antimalarial mefloquine, erythro-2,8-bis(trifluoromethyl)-alpha-(2-piperidyl)-4-quinolinemethanol hydrochloride. Following oral administration of 14C-labeled mefloquine hydrochloride to female mice, the drug was well absorbed and distributed throughout the body. At both 24 and 48 hr after dosing the majority of radiolabel remaining in the body was in the form of parent drug. The major route of radiolabel excretion was fecal after either oral or intraperitoneal administration, with approximately 20% in the urine by 240 hr. The elimination t1/2 of unchanged drug after oral or intraperitoneal dosing was 18.7 and 13.5 hr, respectively, for urine and 14.5 and 15.4 hr, respectively, for feces. The t1/2 of parent drug after oral administration was 17.0 hr for plasma and 18.6 hr for erythrocytes. The erythrocyte concentration of drug was approximately five times the plasma concentration.


Physiological disposition and metabolism of cyclobenzaprine in the rat, dog, rhesus monkey, and man. The absorption, distribution, excretion, and metabolism of 3-(5 H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-1-propanamine (cyclobenzaprine) were investigated in the rat, dog, rhesus monkey, and man. The drug was well absorbed in all species after oral administration. The rat eliminated the drug primarily in the feces, but urinary excretion was predominant in the dog, monkey, and man. The drug was rapidly and widely distributed into rat tissues, highest concentrations being found in the small intestine, lung, kidney, and liver. The drug was highly bound in human plasma. Extensive biliary excretion of the labeled compound was observed in the rat. Major metabolites in the rat were phenolic derivatives but in man the major metabolites were 10,11-dihydroxynortriptyline and cyclobenzaprine glucuronide. Only minor amounts of unchanged drug were present in the urine.


N-Didemethyl-n-propionyl-6,9;9, 12-erythromycin A-spiroketal, a new metabolite of erythromycin ethyl succinate in man. A novel, neutral metabolite of erythromycin was isolated from the urine of a patient treated with erythromycin ethyl succinate. After separation and purification on Sephadex LH-20 column chromatography, the structure of the metabolite was deduced from spectral data to be the propionamide of the didemethylated 6,9;9, 12-spiroketal of erythromycin A.


Enzymatic conjugation and hydrolysis of [18O]isoborneol glucuronide. Introduction of 18O into the acetal linkage of isoborneol glucuronide has been accomplished through the in vitro enzymatic conjugation of 18O-labeled isoborneol by use of immobilized, partially purified, rabbit liver UDP-glucuronyltransferase supplemented with UDP-glucuronic acid. Mass-spectrometric analysis of 18O enrichment in both the aglycon and the intact conjugate made it possible to study both enzymatic conjugation and enzymatic hydrolysis. Analysis by gas chromatography-mass spectrometry indicated that the labeled hydroxyl oxygen of isoborneol is retained during both conjugation and hydrolysis.


A three hours study of the response of plasma corticosterone and of three liver enzymes in rats subjected to stress in the late afternoon or during the morning hours. In adult male rats kept under controlled illumination (lights on from 6 a.m.--6 p.m.) the existence of higher afternoon values of plasma corticosterone ("B") is reconfirmed. Moreover, plasma "B" increases induced by 400 revolutions in Noble-Collip drums or immobilization for 2.5 h were found substantially less pronounced if stressor application took place at 4 p.m. rather than 8 a.m. so that essentially no difference between morning and afternoon poststress peaks was observed. Also the response to 0.05 mg ACTH i.p. was smaller in the afternoon experiments. Additionally three liver enzymes the activity of which is increased by corticosterone released under conditions of stress were also studied. Higher afternoon activity of liver tyrosine aminotransferase (TAT) and tryptophan pyrrolase (TP) was observed in spite of fasting from 8 a.m. imposed to the animals taking part in the afternoon experiments. The activity of phosphoenolpyruvate carboxykinase (PEPCK) was also increased in the afternoon probably because of the food deprivation. Immobilization-induced increase in the activity of all three enzymes showed a similar blunting as plasma "B" response, so that no differences between morning and afternoon were found in peak poststress values. In conclusion, the low plasma "B" response of rats at a time when the animals begin the active part of their day seems to have important metabolic implications.


beta-Galactosidases in mouse duodenal mucosa. beta-Galactosidase activity, in fetal mice, first appears at 16 days of gestation and has a pH optimum of 4. In postnatal development the enzyme activity of cell homogenates tends to show bimodal pH at 4 and 5.6. There are two molecular forms of the enzyme, separable both by molecular-sieve chromatography and electrophoresis. One of the molecular forms of the enzyme is active over a wider range of pH (3.2-6.2) and has half as much activity at 5.6 as it does at 4. This isoenzyme is continuously present in both fetal and postnatal stages. The second isoenzyme first appears at birth, is active over a narrower range of pH (4.6-6.2) and inactive at PH 4. The bimodal pH optima observed in postnatal stages in the cell homogenates, appears to be due to the combined activity of the two molecular forms. In isolated brush border membranes, isoenzyme 2 is the only one present. The other organelles (mitochondria, microsomes, lysosomes, nuclei and cytoplasm) have variable proportions of both isoenzymes, as indicated by the activity ratio at pH 4/5.6.


Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes. The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila  molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components.  No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).


Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium. Phosphorylation of the calcium-transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 degrees C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees C and pH 6.2 is in the range of 3.6--4 nmol/mg protein. Half-maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1--0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1--0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.


The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica. Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.


Purification and properties of a new restriction endonuclease from Haemophilus influenzae Rf. Haemophilus influenzae Rf 232, showing the phenomena of restriction and modification, contains an endonuclease that inactivates in vitro the biological activity of DNAs lacking the strain-specific modification. This specific restriction endonuclease has been purified to near homogeneity by a procedure that includes DNA-agarose chromatography. This highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S-adenosylmethionine. The enzyme seems to cleave DNA at well-defined sites, since it produces a specific pattern of bands upon agarose gel electrophoresis. The enzyme has no ATPase activity. A methylase activity is observed in the course of the endonucleolytic reaction, which probably protects some of the DNA sites from cleavage.


Calcium-induced inactivation of microtubule formation in brain extracts. Presence of a calcium-dependent protease acting on polymerization-stimulating microtubule-associated proteins. Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.


Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen. A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.