Delayed fluorescence from Rhodopseudomonas viridis following single flashes.

Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studies using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature.  The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll.  The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P+) of the primary electron-donor (P) and the photoreduced form (X MINUS) of the primary electron-acceptor.  This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P+ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P+X minus, and (c) upon transfer of an electron from X minus to a secondary acceptor, Y.  (2) Under conditions that prevent the reduction of P+ by cytochromes and the oxidation of X minus by Y, the decay kinetics of delayed fluorescence are identical with those of P+X minus, as measured from optical absorbance changes.  The main decay route for P+X minus under these conditions has a rate-constant of approximately 10-3-s-minus 1.  In contrase, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P+X minus returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s-minus 1, at 295 degrees K and pH 7.8.  The decay kinetics of P+X minus and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal mol-mol- minus 1.  We conclude that the main decay route involves tunneling of an electron from X minus to P+, without the promotion of P to an excited state.  Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare.  The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P+X minus results in increases of both the energy and the entropy of the system, by 16.6 kcal-mol- minus 1 and 8.8 cal-mol- minus 1-deg- minus 1.  The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.


On the stimulation of the light-induced proton uptake by uncoupling aminoacridine derivatives in spinach chloroplasts.

1. Light-induced proton uptake by spinach chloroplasts is enhanced several-fold by 9-(4-diethylamino-1-methylbutylamino)-6-chloro-2-methoxyacridine (atebrin).  This stimulation does not depend on the chlorophyll concentration.  The amount of extra protons taken up in the presence of atebrin is determined by the pKa values of atebrin and the pH of the incubation medium.  2. Both the stimulation of the proton uptake and the maximal binding capacity for atebrin is sensitive to uncouplers.  However, the ratio of bound to free atebrin does not depend on the presence of uncoupler up to the saturating atebrin concentration.  3. From simultanious kinetic measurements of atebrin fluorescence and proton movement it seems that after binding of the completely protonated atebrin the dye and the protons can move separately.  This can also be inferred from the spectral behaviour of atebrin in illuminated chloroplasts.  4. The stimulation of the proton uptake by atebrin does not depend on the presence of salts in the incubation medium.  However, the 'saturating' atebrin concentration increases strongly with increasing salt concentration in the medium.  5. It is concluded that the interaction of atebrin and other acridines with energized chloroplasts most likely occurs at the level of the membrane proper.  6. It is proposed that uncoupling by atebrin is a consequence of the creation of a high proton activity at the periphery of the thylakoid membrane, which opposes a proton gradient across the membrane.  The uncoupling by atebrin is not of the protonophoric type according to this mechanism.


A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria.

Generation of membrane potential (delta psi) and transmembrane pH difference (delta pH) was studied in PPi-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport.  The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of delta psi, while an atebrin response is an indicator of delta pH.  The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by delta psi and delta pH.


Phospholipase A activities in rat placentas of 14 days' gestation.

1. Homogenates and subcellular fractions of placentas obtained from rats on the 14th day of gestation were assayed for enzymes characteristic of various subcellular organelles. Based on these assays it seemed the placental lysosomes (acid phosphatase activity) were distributed equally among the subcellular particulate fractions and that the cytosol was not contaminated by mitochondria or microsomes. 2. The placental preparations were assayed for phospholipase A activity using 2-[14-C] phosphatidylethanolamine. We found phospholipases A with pH optima of 4.0, 7.0, and 8.5. These enzymes had Ca-2+ requirements and subcellular localizations similar to those reported for adult rat liver (Waite, M., Scherphof, G.L., Boshouivers, F.M.G. and van Deenen, L.L.M. (1969) J. Lipid Res. 10, 411.) 3. The majority of the placental phospholipase A activity was found in the cytosol. We think this activity represents enzyme(s) solubilized from the plasma membranes of placental cells and speculate that this enzyme may be active in a placental transport mechanism.


Metabolism of alkyldihydroxyacetone phosphate in rat brain.

Alkyldihydroxyacetone-P is the first detectable product in the biosynthetic pathway for ether-linked glycerolipids that eventually leads to the formation of ethanolamine plasmalogens, a major constituent of myelin. During early postnatal development, the specific activity of NADPH2:alkyldihydroxyacetone-P oxidoreductase in microsomes from rat brain is maximum at 4-5 days after birth, the time when the specific activity of the enzymes that synthesize alkyldihydroxyacetone-P also peaks. For the oxidoreductase assay, we developed a thin-layer chromatographic method that separates alkyldihydroxyacetone-P as the dinitrophenylhydrazine derivative from its reduced product (alkylglycerol-P), with excellent resolution. Phosphohydrolases associated with brain microsomes exhibit optimal pH maximums at 5.2-5.6 and 7.5-7.8 for all three substrates tested -- alkyldihydroxyacetone-P, alkylglycerol-P and alkylacylglycerol-P. Alkylglycerol-P was most readily dephosphorylated under all experimental conditions. The enzyme(s) that dephosphorylates alkyldihydroxyacetone-P and alkylglycerol-P have similar properties with respect to Mg-2+ or EDTA; with both substrates, Mg-2+ had no effect and EDTA was highly stimulatory. In contrast, EDTA strongly inhibited the dephosphorylation of alkylaclglycerol-P and although Mg-2+ (1 mM) appeared to be required for optimal activity, higher levels inhibited the reaction.


Glycosphinoglipid beta-galactosidases of cultured mammalian cells. Characterization of the enzymes from mouse cell line lmtk and human Lesch-Nyhan fibroblasts.

Evidence is presented for the existence of three distinct mammalian glycosphingolipid beta-galactosidase responsible for the hydrolysis of galactosylceramide, lactosylceramide and GM1 gangliside, respectively. Activity toward the (L-3-H)galactose-labeled substrates differed with respect to pH optimum, thermostability, effect of NaCl and inhibition by glycosides and related glycosphinglpids. Comparison of these enzymic acitivites in cultured mouse cell line LMTK- and human beta-galactosiddases could probably be detected in future experiments with somatic cell hybrids (formed by the fusion of these two cell strains by specifically inhibiting activity of mouse origin.


Intracellular pH of Thermoplasma acidophila.

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 degrees C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.


Phosphate transport in Neurospora. Derepression of a high-affinity transport system during phosphorus starvation.

In addition to the constitutive, low-affinity phosphate-transport system described previously, Neurospora possesses a second, high-affinity system which is derepressed during phosphorus starvation. At pH 5.8, System ii has a K1/2 of about 3muM and a Jmax of 5.2 mmol/1 cell water per min. System ii reaches maximal activity after about 2 h of growth in phosphorus-free minimal medium. Its formation is blocked by cycloheximide and, once made, it appears to turn over rapidly. Addition of cycloheximide to fully derepressed cultures results in the decay of System ii with a t1/2 of 14 min, very similar to the turnoacteriol. 95, 959-966) for tryptophan transport in Neurospora. Thus, these transport systems appear to be regulated by a balance between synthesis and breakdown, as affected by intracellular pools of substrate or related compounds.


Observations on the binding of adenosine 3':5'-monophosphate to cell membrane fragments from ox cerebral cortex.

Microsomal or synaptosome membrane fragments from ox brain bind cyclic AMP with a pH optimum of 7.0. Scatchard analysis shows the presence of at least two binding sites. Cyclic GMP and cyclic IMP only inhibit binding at concentrations 5000 times that of cyclic AMP and even higher concentration ratios of ATP and AMP have no effect. Membrane fragments saturated with cyclic [3-H]AMP lost less than 7% of bound nucleotide on incubation at 0 degrees C for 45 min but lost 25 % in the same period in the presence of 10 muM non-radioactive cyclic AMP.


Transcription and division inhibitors in the medium of stationary phase cultures of the slime mold Dictyostelium discoideum.

When cell-free medium of a stationary phase culture is added to an exponentially multiplying culture, it blocks further cell division and depresses the level of nuclear transcription. Furthermore, when cell-free medium of a stationary phase culture is added directly to an in vitro transcription system containing nuclei isolated from exponentially multiplying amebae, it completely inhibits "late" incorporation (20-60 min reaction time), which is predominantly alpha-amanitin resistant (ribosomal-like). The stationary phase medium however, affects neither "early" alpha-amanitin resistant incorporation (0-20 min reaction time) nor alpha-amanitin sensitive incorporation. Inhibition of late in vitro transcription is not due to a nucleotidase, nor is it due to changes in the pH, ionic strength, or osmolarity of the reaction mixture.


Purification and characterization of an endonuclease from calf thymus acting on irradiated DNA.

An endonuclease acting on DNA exposed to ultraviolet light or gamma-rays has been extensively purified from calf thymus. The enzyme has a pH optimum at pH 7.0-7.5, acts with equal efficiency in the presence of EDTA or divalent cations (Mg-2+ or Ca-2+), is inhibited by NaCl and tRNA and is inactivated by incubation at 50 degrees C. Its molecular weight, determined by Sephadex chromatography or sodium dodecylsulfate gel electrophoresis, is approx. 30 000. The enzyme catalyzes the formation of breaks with 5'-phosphate termini in double-stranded DNA irradiated with ultraviolet or gamma-rays. It does not act on unirradiated DNA or denatured DNA. Since in all these properties the enzymatic activity on ultraviolet- and gamma-irradiated DNA behaved similarly and since the two activities cochromatographed in all systems used during purification, we conclude that they are associated with the same protein. The site of action of the enzyme in ultraviolet-irradiated DNA is a photoproduct other than pyrimidine dimers. Such a photoproduct can also be induced by irradiation of the DNA in vivo, i.e. within the cells.


The presence of collagenase in collagen preparations.

The frequently observed instability of neutral salt solutions of native collagen extracted from various sources and partially purified by standard procedures has been studied by disc electrophoresis in polyacrylamide gel and by electron microscopic examination of segment long spacing crystallites. The phenomenon has revealed time and temperature dependency, pH optima near neutrality, and inhibition by sodium EDTA and serummin addition, collagen breakdown has been found to be quantitatively related to the state of aggregation of the substrate, being more marked in reconstituted collagen gels than in collagen in solutionma typical pattern of animal collagenase degradation of native collagen into two fragments designated as TC-A and TC-B has been observed under certain conditions. It is concluded that the degradation of native collagen in neutral salt solution is due to a specific collagenase, and that this enzyme probably remains bound to collagen throughout the process of extraction and partial purification. Experiments with gelatin suggest that, in addition to collagenase, a nonspecific proteolytic activity may also be present in collagen preparations.


[Study of the cell walls of yeasts Rhodotorula. VI'nfluence of 2-hydroxybiphenyl on phosphatase activities of Rh. rubra (author's transl)].

The inhibition of acid phosphatase activity observed after culture of Rh. rubra in phosphate rich mediums is raised by the culture of this yeast in presence of 2-hydroxybiphenyl (OHph2). The cell wall alkaline phosphatase activity was inhibited by this derivative; When cultivated with OHph2 an intra and a more extracellular acid phosphatase activity appeared. The comparative studies of the two extracellular acid phosphatases secreted in the medium with or without the OHph2 show they have similar characteristics. They are eluated at the same time from Sephadex  G-200, DEAE- and CM-cellulose columns, and have the same Km. They are both glycoproteins, with the sugars forming the polyose fragment identical, but the enzyme secreted in the medium containing the OHph2 contains less sugar than the one secreted in the medium without OHph2. The appearance of this acid phosphatase activity was attributed to the alteration of the membrane glycosylating systems or to the important ultra structure modifications of the cell wall of Rh. rubra when this yeast is cultivated with OHph2.


The interaction of cobalt (II) complexes with bovine apocarbonic anhydrase B.

The second-order rate constants for interaction of a number of cobalt(II) complexes (including the aquated ion) with bovine apocarbonic anhydrase have been measured at pH equal to 7.5, 25 degrees C and I equal 0.1 M. The ease of entry of cobalt decreases, in general, as the number of its coordinated watersdecreases. The highest complexes (bis or tris) do not react. The Mono cobalt(II) -8-quinolinol-5-sulfonate and, particularly, 8-quinolinol complexes react very much more rapidly with apoenzyme than does Co(H20)6-2+ ion. The results are discussed.


Kinetics of iron and copper catalysis of ascorbate oxidation.

The effects of oxidant, pH and ligands on iron- and copper-catalyzed ascorbate oxidation have been examined. The formation of the catalyst-substrate complex is affected by pH, whereas oxidant affects its breakdown. With copper-ion catalysis, ligands inhibit competitively. With iron catalysis, on the other hand, for a series of aminopolycarboxylic ligands at neutral pH, formation of catalyst-substrate is favored by ligands which form more stable iron complexes. Decreased rates caused by changes in metal environment (ligand or pH) may result for competing activities (e.g., catalase activity competing with peroxidase activity). Evidence for a ternary complex (catalyst-substrate-oxidant) is presented.


Quantitative magnetic resonance studies of manganese uptake by mitochondria.

The uptake of the paramagnetic ion manganese by rat liver mitochondria is studied by electron paramagnetic resonance (EPR) spectroscopy. Emphasis is placed on: (a) obtaining accurate EPR quantitation of intramitochondrial manganese fractions previously described (Gunter, T. E., and J. S.Puskin, 1972, Biophys. J. 12:625) (b) establishing competition for intramitochondrial binding between one of these fractions and calcium, (c) demonstrating the effects of substrate and ATP concentrations on each fraction observed through EPR, and (d) demonstrating the effect of inorganic phosphate (Pi) concentration and pH on each fraction.


The self-association of ATP: thermodynamics and geometry.

The concentration and temperature dependence of the self-association of ademosin-5'-triphosphate (ATP) in aqueous solution was studied by means of ultraviolet absorption spectroscopy and circular dichroism (CD). Of several possible models, a model was  indefinite linear self-association, in which each step has the same equilibrium constant, describes the data best. The two different methods lead within experimental error to the same thermodynamic parameters. At pH 8.7, IN 1 M Tris and 0.5 M 7gCl-2, we find deltaH-0 equals -5.1 kcal/mole and deltaS-0 equals -13.0 e.u. These values do not differ much from those found for the self-association of uncharged bases and nucleosides in  aqueous solution. The CD spectrum that results from the self-association is conservative and quite similar in shape to that observed for some stacked dinucleotides: it is interpreted as a first approximation within the framework of the exciton model.


Nuclear Magnetic resonance quadrupole relaxation studies of chloride binding to the isolated hemoglobins from trout (Salmo irideus).

NMR studies of chloride binding to the main components of trout blood, Hb Trout I and Hb Trout IV, indicate that although the affinity of chloride is high for both hemoglobins, the characteristics of the binding process are markedly differnet. In Hb Trout IV chemical exchange at the chloride binding site(s) is fast and quadrupole effects determine the linewidth; chloride binding has a definite pH dependence, but there is no significant oxygen linkage. In contrast Hb Trout I represents a unique case of slow chemical exchange, which may depend on unusual stereoche mical characteristics of the chloride binding site; chloride binding is pH independent, but shows a significant oxygen linkage, which may be attributed to changes of the lifetime of chloride at the binding site. The chloride binding properties displayed by Hb Trout I and IV have been compared with those of normal and modified human hemoglobins and discussed in terms of the structural differences in the C- and N-terminal regions of the alpha- and beta-chains.


Cation bindint to alpha-sl-casein B. A comparison of electrostatic models.

System characteristics which determine calcium binding to and subsequent proton release from alpha-sl-casein B are reported at pH 6.6 and [Na-plus] equal to 0.04, 0.0, and 0.16M. Values of protein solvation, G, site bound calcium, Ca,S, and net monomer charge, Z, permitted distributed charge models to be constructed. The models examined proved inadequate in that it was impossible to keep the dielectric constant, D, within acceptable limits and/or predict measured proton release. Three discrete charge models were constructed. At D equals 4, all three gave good agreement between predicted and experimental data as Ca, S increased. The known amino acid sequence was used to make rodlet models for the whole molecule andfor just the phosphate-containing acidic peptide portion. A comparison of these shows the electrostatic dominance of the acidic peptide and suggests that the electrostatic environment for the remainder of the binding sites is essentially constant as Ca, S increases during addition of calcium ion. The third discrete charge model bends the acidic peptide rodlet into a torus. In this case, data were matched with less assumed bond strain under conditions of high molecular charge than with the other two models. This indicates that conformation and association may be important factors to consider when constructing discrete charge models to calculate electrostatic free energy.


[The ultrastructure of the parietal cells of the stomach and their functional activity].

Submicroscopic changes in the parietal cells of the fundus were compared with the data of spot-film intragastric pH-metry of the same mucosal areas in 18 patients treated at the hospital in the department of therapeutic food deprivation. There were seen no changes in the ultrastructure of the parietal cells after a brief (36-hour) fasting when the pH of the mucous membrane failed to differ from the normal. With increased duration of food deprivation there occurred a gradual alkalinization of the gastric secretion and the pH of the gastric wall shifted from 2.02 after a 3-day food deprivation to 4.8-5.0 after fasting for 20 to 30 days. In accordance, there was a change in the ultrastructure of the parietal cells consisting in flattening and subsequent disappearance of tubulovesicles, a reduction of the lumina of the intracellular channels and shortening of microvilli. The results of comparison of the submicroscopic changes with the data of pH-metry indicated that such submicroscopic shifts corresponded co depression of the functional activity of the parietal cells.


[The role of mast cells in vascular permeability disorders in rats subjected to immobilization stress].

Disturbances of vascular permeability were studied by the "vascular labeling" technique in the mesentry during the 24-hour immobilization of rats. Administration of dimebolin (an antihistaminic preparation) decreased the number of labeled vessels and labeling intensity. This effect was expressed in the presence of mast cells only and was accompanied by the mast cell degranulation. The authors suppose that the mast cells contain a substance preventing the disturbance of vascular permeability and released during degranulation. Such substance might be heparin. Experiments showed that small doses of heparin failed to produce such effect. These results allowed one to conclude that mast cells played a double role in the mechanisms of disturbance of vascular permeability during immobilization--the damaging (by the action of histamine and serotonine) and the protective (by the released heparin) action.


[Thymidylate synthetase in the antimesometrial and mesometrial portions of decidual tissue under normal conditions and following application of the antifolate preparation chloridine].

Specific activity of thymidylate synthetase (TMS) under normal conditions and after the action of pyrimethamine was investigated in antimesometrial (A) and mesometrial (M) parts of rat decidua. On the 9th-11th days of pregnancy specific activity of TMS in the A part proved to be higher than in the M part of decidua, and the former one reached its maximum by the 10th day. Specific activity of TMS in the M part decreased gradually from the 9th to 11th days of gestation. Pyrimethamine, applied on the 9th day of pregnancy caused a wave-like increase in the specific activity of TMS in the A part; this elevation was not so pronounced in the M part; A study of the physico-chemical properties of TMS from the decidua and the embryos of rats showed that the enzyme had a molecular weight of 58000, an optimal pH of 6.9, and could be quickly inactivated by heating.


The effect of platelet concentrate storage temperature on adenine nucleotide metabolism.

The effect of platelet concentrate storage temperature (4 degrees C versus 22 degrees C) on platelet adenine nucleotide metabolism was studied. In general, levels of platelet ATP and ADP, the release reaction, and the metabolis nucleotide pool were best preserved for 72 hr by storage of concentrates at 4 degrees C. Storage of concentrates for 72 hr at 22 degrees C was occasionally associated with a pH decrease to less than 6.0, which is incompatible with platelet viability. When the pH fell below 6.0, there was a marked deterioration of platelet adenine nucleotide levels and the release reaction. The results for concentrates stored at 22 degrees C, with a final pH above 6.0, were not inferior to the results for those stored at 4 degrees C. The pH remained above 7.0 in all concentrates stored at 4 degrees C. The pH changes of platelet concentrates stored at 22 degrees C could not solely be attributed to platelet count, red cell count, or bacterial contamination. Storage at both temperatures was associated with conversion of ATP in the metabolic adenine nucleotide pool to hypoxanthine.


Activation of histidine decarboxylase by H2-receptor blockade: mechanism of action.

1 Treatment with histamine H2-receptor antagonists, which inhibit basal acid secretion was found to activate rat stomach histidine decarboxylase. At the same time the serum gastrin concentration was greatly increased. 2 In antrectomized rats neither the enzyme activity nor the serum gastrin concentration was affected by the treatment. 3 In analogy with previous observations on other inhibitors of acid secretion we suggest that the H2-receptor antagonists stimulate gastrin release through their effect on acid secretion and that the raised serum gastrin level is responsible for the enzyme activation.


Influence of histamine H1- and H2-receptor blockers on sympathetic vasodilator and vasoconstrictor responses in canine paw.

1 Vasodilator responses to histamine, bradykinin and sympathetic nerve stimulation were elicited in the perfused paw of dogs treated with bretylium (15-20 mg/kg) and atropine. The H2-receptor blocking agent, burimamide, when administered in the dose of 5 mg/kg intravenously and 4 mg intra-arterially did not depress significantly these vasodilator responses. The subsequent administration of tripelennamine in the dose of 2.5-5 mg/kg intravenously and 4 mg intra-arterially produced a significant blockade of the response to histamine and reduced the sustained vasodilator response to nerve stimulation. 2 In guanethidine-treated dogs, tripelennamine administered in the same dose following burimamide produced a blockade of the response to histamine comparable to that in the bretylium experiments, but decreased only the sustained vasodilator response to stimulation at 1 Hz. When the order of administration of the antihistamines was reversed in another group of guanethidine-treated dogs, tripelennamine had only a slight blocking effect on the response to histamine and did not affect the responses to nerve stimulation. Burimamide exerted a significant blocking effect on the response to histamine, but not to nerve stimulation. Another H2-receptor blocking agent, metiamide, when given after tripelennamine, also had a marked blocking effect on the response to histamine and almost abolished the vasodilator response to 4-methylhistamine, an H2-agonist. Nevertheless, even in the presence of this profound histamine blockade, the sustained vasodilator response to nerve stimulation remained unchanged. 3 In another group of experiments vasoconstrictor responses to exogenous noradrenaline and sympathetic stimulation were initially depressed by burimamide and later returned to control values. Tripelennamine increased these responses by its uptake blocking action. 4 It is concluded that the sustained vasodilator response is not antagonized by a specific antihistaminic action. The decrease in the sustained vasodilator response produced by antihistamines is produced attributable to potentiation of a residual adrenergic vasoconstricotr effect not completely blocked by bretylium.


EEG features of depressive and schizophrenic states.

A 25-year-old female patient with a psychotic illness lasting three months in which schizophrenia and depressive episodes occurred was studied by serial EEGs. These were coded, masked and rated in a set way without knowledge of the clinical state. It was found that the schizophrenic state was characterized by a decrease in the amount and poor organization of alpha activity, as well as the occurrence of paroxysmal phenomena in particular a typical spike and wave. In the depressive episode, alpha activity was prominentand only scanty paroxysmal features were seen. Using these criteria a series of EEGsfrom psychiatric patients were assessed 'blind' with a view to separating them into either a schizophrenic or a depressive category. This proved possible in 17 cases (9 depressive and 8 schizophrenic). In only two was the categorization completely misjudged. These results suggest that further detailed study of the EEG, possibly with computer analysis, combined with assessment of the behavioural state might not only yield useful diagnostic information but also lead to a better understanding of the underlying neurophysiological basis of certain mental disorders.


Acute reactions to urographic contrast media.

A prospective study of 3509 consecutive patients examined by excretion urography has been conducted to assess the incidence and significance of the untoward effects of urographic contrast media. Four compounds were used in doses containing 160 to 500 mg iodine/kg body weight. Toxic effects, arm pain, and allegic reactions were assessed separately, while the remainder were classified according to the influence of each reaction on the investigation and the need for treatment. From the results and a review of the literature we conclude that when there is a clear clinical indication for excretion urography a dose of contrast medium containing up to 600 mg iodine/kg body weight should be injected rapidly. Prophylactic antihistamine treatment and pretesting should be abandoned. Special care is needed for small infants and the lederly and for patients with renal or hepatic failure, myeloma, heart disease, or a history of previous major reaction. Full resuscitation facilities must always be available.


Pulmonary function in asthmatic patients in remission.

Thirty-five asthmatic patients (average age 28 years) who attended a pulmonary function laboratory when their mean ratio of forced expiratory volume in one second: forced vital capacity was 81 per cent (within the normal range for their age group) had arterial hypoxaemia and hypocapnia. These were probably secondary to lung hyperinflation and pulmonary ventilation/perfusion imbalance. The pulmonary abnormalities of bronchial asthma are not always detected by simple spirometric tests and the results of such tests should be interpreted cautiously.


Changes in haemoglobin binding curve and oxygen transport in chronic hypoxic lung disease.

Direct measurements of the factors determining blood oxygen transport in 10 patients with chronic hypoxic respiratory failure led to the conclusion that wide differences in the position of their oxygen binding curves, due to spontaneous differences in red-cell 2, 3-diphosphoglycerate, had little effect on oxygen delivery to the tissues, as assessed by the mixed venous oxygen tension when they were breathing air. This result arises from the shape of the oxygen binding curve. A drug which could shift the curve to the right would help tissue oxygenation in cardiogenic and other forms of shock, when a low cardiac output can not be improved though arterial blood can be well oxygenated.


Functional and biochemical aspects of catecholamine metabolism in brain under hypoxia.

Animals exposed to 6% oxygen showed a partial inhibition of the rate of tyrosine hydroxylation and a blockade of the conditioned avoidance response. The behavioral disruption was suggested to result, at least in part, from a dopaminergic disturbance, since the behavior was restored by the administration of DOPA or apomorphine but not by 5-hydroxytryptophan. Biochemical data showed a selective retardation in brain dopamine (DA) disappearance after synthesis inhibition. Methoxytyramine formation was markedly retarded. Analysis of this effect indicated that the decreased DA turnover was related to the release of transmitter rather than to an effect on catechol-O-methyltransferase (COMT) or monoamine oxidase (MAO). In addition, there was evidence for a partial inhibition of MAO activity by hypoxia as well as a decreased activity of dopamine-betahydroxylase. It was concluded that the disruption of behavior was related to decreased dopaminergic receptor activation and that decreased synthesis played at most a contributory role.


The effect of pH and blood gas correction in DPG and plasma potassium content of stored blood.

During storage of CPD preserved blood, red cell DPG decreases and plasma potassium increases, the changes becoming more marked with increasing duration of storage. Correction of the blood gas and pH alterations of stored blood decreases plasma potassium and increases red cell DPG, but they do not return to normal except in blood stored less than seven days. There is a highly significant correlation between increasing red cell DPG and decreasing plasma potassium as the pH and blood gases of stored blood are restored to physiological range.


Evidence for NAD nucleosidase in rabbit-liver lysosomes.

A method is described for the isolation of secondary lysosomes from homogenates of rabbit liver; The uptake of Triton WR-1339 by rabbit-liver lysosomes when administered by intraperitoneal injection was used to decrease the density of secondary lysosomes. Lysosomal fractions prepared by this method contain an NAD nucleosidase (NAD glycohydrolase, EC 3;2.25), an enzyme which has previously been considered to be associated with other subcellular fractions. The enzyme has maximum activity at pH 6 and cleaves both NAD and NADP. It is inhibited by nicotinamide (Ki equals 4.5 mM) and by HgCl2. Both nucleosidase and 2'-nucleotidase show in-vitro latency typical of lysosomal acid hydrolases. Rabbit-liver plasma-membrane fractions were isolated which contained most 5'-nucleotidase but relatively little nucleosidase, whereas rabbit liver lysosomes contain both 5'-nucleotidase and nucleosidase enzymes but little adenyl cyclase.


Structural studies of staphylococcal protease. I. Spin labelling of the active site and a comparison with other proteases.

Staphylococcus aureus protease has been spin-labelled at the active-site serine residue with the monocyclic-phosphorus spin label (MSL), 1-oxyl-2,2,6,6-tetramethyl-4-peperi-dinylethylphosphorofluoridate. The electron paramagnetic resonance (E.P.R.) sbectra of the protease in different buffers at various pH's have been analyzed and compared with those of trypsin, subtilisin BPN', and alpha-chymotrypsin under identical conditions. In a given buffer, the shape of E.P.R. signals of spin-labelled staphylococcal protease is unaffected by pH changes except below pH 4.0, at which a gradual loss of conformational integrity of the active site occurs. In bicarbonate buffer and particularly in acetate buffer, the mobility of the label is much more restricted than in phosphate buffer or in potassium chloride solution. The implications of this finding are discussed in terms of a model whereby the label is able to orient towards two different but adjacent regions of the active site. The relative population of the label in each of these orientations is believed to be buffer-dependent. An attempt to correlate the shape of the te.p.r. signals with the pH values of maximal proteolytic avtivity of the enzyme is also presented. These results show that to obtain meaningful information from a comparative spin label study of the geometry of the active site of serine proteases, particular care should be exercised to assure that the different proteases experience identical conditions of pH, buffer, and temperature.


Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.


Protein kinases in rat testes: evidence for different fractions of the enzyme.

Protein kinase activity of rat testis homogenate was separated into five fractions by means of pH 4.8 acidification and DEAE-cellulose chromatography. The five fractions showed a peculiar pattern of activity and cAMP dependency with the substrates used: casein, protamine, histone mixture, arginine-rich histone, lysine-rich histone, and phosvitin. The casein-sepharose substrate affinity column separated two fractions from the pH 4.8 precipitate. Peak number one phosphorylates histone preferently and is cAMP-dependent, while peak number tow has a strong affinity toward casein as substrate and is non cAMP-dependent.


The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate.

The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified. The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography. On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000. Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme. The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions. Sulfate and phosphate ions are inhibitory to the enzyme. Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators. The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway. Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators. Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu. Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation. The effectors act by changing the Michaelis constant for the substrates. Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate. The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.


Survey of antibiotic residues in Canadian slaughter animals.

Kidneys and urine of cattle, swine, sheep and chickens were tested for bacterial growth inhibitors using Bacillus subtilis and Sarcina lutea as test organisms. Results were as follows: 211 beef kidneys four positive, 611 swine kidneys five positive, 27 sheep and 120 chicken kidneys all negative, 2108 beef urine 76 positive, 2409 swine urine 186 positive, 176 sheep urine 17 positive. Strongest reactions were obtained with B. subtilis on phosphate buffered pH 6.0 plates. Larger zones were produced by urine from injected animals than by tissue samples.


The use of a travelling ophthalmic technologist in a remote, sparsely populated region.

An attempt has been made to meet the ophthalmological needs of the population of the 500,000 square mile Mackenzie Zone of the Canadian Northwest Territories by the use of a well trained ophtalmic technologist who travels to each of the small, widely separated communities. He carries out the necessary refractions and screens patients selected by the local nurses for ocular disease and/or motility problems for referral to the ophtalmologist in charge. Patients requiring treatment or further investigation are referred to the supervising ophtalmologist who will provide instructions by phone or radio, will see them on a subsequent visit, or if indicated, will have them evacuated to the well equipped treatment centre in Yellowknife. Some of the advantages of this programme are as follows: 1. Communities are visited on a regular schedule with reasonable frequency by the same personnel. 2. The skills and training of the ophthalmologist are more effectively used by enabling him to spend most of his time at the centre where adequate medical and surgical facilities are available. 3. Standardized, centralized records are kept which are identical to those in patient's home community. It is suggested that the use of well-trained paramedical personnel may be of significant value in the provision of adequate medical care to widely dispersed population groups in the far north.


Cellular immunity to xenogeneic corneal grafts in rabbits.

We wished to determine whether a small orthotopic corneal xenograft could sensitize a host systemically. Ten rabbits were grafted with interlamellar bovine cornea and pre- and postoperative blood samples tested by the H3 thymidine incorporation test (micro-method). The leucocyte migration inhibition test was applied in 3 animals. A skin test was performed on all rabbits in the 4th postoperative week. The challenging antigen used  was a homogenate of pooled bovine corneas. Clinically and histologically each rabbit showed a characteristic corneal graft reaction. Where performed, leucocyte migration inhibition tests were positive after grafting as were all skin tests. The H3 thymidine incorporation test did not detect specific systemic sensitization although increased numbers of DNA synthesizing cells were present in the blood during the graft reaction. We conclude that a bovine corneal xenograft can induce systemic sensitization in a rabbit host.


Degradation of feather keratin by culture filtrates of Streptomyces fradiae.

Treatment of milled defatted capon feathers with filtrates of Steptomyces fradiae ATCC 10745 that had been concentrated 20 times showed that the most keratin degradation in 0.5h occurred at pH 8.0 and 60degrees C. If assay time was extended, however, adecrease in reaction rate occurred and appeared to result from keratinase instability at elevated temperatures. Exposure of commercial feather meal to unconcentrated culturefiltrates for 15 h produced greatest degradation at 50 degrees C.


Differential isolatoion of Pythium species from soil by means of selective media,temperature, and pH.

Pythium aphanidermatum, with an optimum temperature for growth at 35C, grew welland was readily isolated from soil on pimaricin-vancomycin medium (MPVM) when incubated for 24h at 38-40C. The pH of the medium affected recovery; maximum numbers developed above pH 6.0. Other Pythium spp. were recovered on MPVM at 20-25C, but were excluded by incubation at 38-40C. These Pythium spp. included P. ultimun, P. paroecandrum, P. irregular, P. mamillatum, and an unidentified Pythium sp. These species grew well and were readily siolated from soil on gallic acid medium (GAM) when incubated for 24-8h at 20 C.P. aphanidermatum and P. myriotylum grew from mycelium on GAM, but their oospores did not germinate nor could they be isolated from soilon this medium. P. myriotylum grew well on MPVM, but was only rarely isolated, evenfrom soils with known high potential for disease caused by P. myriotylum. Propagules of Pythium were enumerated by a plate-dilution frequency method or by a smearplateethod is valuable for studies on the ecology, survival, and inoculum potential in soils with mixed populations of P. aphanidermatum and other Pythium spp.


N-6-(delta-2-isopentenyl) adenosine: hydrolysis by a mucleosidase isolated from Lactobacillus acidophilus cells.

A nucleosidase activity has been isolated from Lactobacillus acidophilus which rapidly hydrolyses N-6 (delta-2-isopentenyl) adenosine to its corresponding base, N-6(delta-2-isopentenyl) adenine. The activity can be distinguished from the spleen exzyme (EC. 2.4.2.1), a purine nucleoside transferase, on the basis of its substrate specificity, electrophoretic behavior, and nondependence on phosphate. The bacterial enzyme hydrolyzes both inosine and isopentenyl adenosine, giving Km values of 63.3muM and 177 muM respectively. The presence of this enzyme in bacteria counts for the rapid conversion of the parent nucleoside to isopentenyl adenine, which has been observed in these cells. The enzyme thus assumes importance as one of the catabolic activities available to the cell for metabolizing the cytokinin, N-6-(delta-2-isopentenyl) adenosine.


Laboratory diagnosis and monitoring of rheumatologic diseases.

Improved laboratory investigative techniques now foster an increased clinical interest in and awareness of the rheumatologic disease. This review is a discussion of the relevance of laboratory tests used in the more common rheumatologic disorders and of their role in both the diagnosis and assessment of these diseases from the standpoint of the practising clinician.


Quantitative precipitin studies on the specificity of an extract from Tridacna maxima (Röding).

Haemolymph from the clam Tridacna maxima precipitated with purified H-blood-group substances, Helix pomatia galactogen, and pneumococcus type XIV polysaccharide. Although gel diffusion, gel electrophoresis, and inhibition experiments indicated that only a single precipitating lectin was present in the haemolymph, quantitative precipitin and haemagglutination results suggested that a second agglutinin with anti-H-like specificity was also present. Evidence obtained from hapten inhibition experiments indicated that the precipitin that reacts with pneumococcus type XIV polysaccharide can be inhibited by a number of simple sugars. Of the compounds tested, 2-acetamido-2-deoxy-D-galactose was the best inhibitor of precipitation with pneumococcus type XIV polysaccharide and of haemagglutination with human erythrocytes, but the inhibition experiments showed that the extract was also markedly inhibited by D-galactosamine hydrochloride, D-galactose, lactose, and p-nitrophenyl beta-D-galactopyranoside. The latter compound was more active than its parent sugar, which was in turn a more potent inhibitor than p-nitrophenyl alpha-D-galactopyranoside. Melibiose, raffinose, and stachyose, compounds which each contain terminal alpha-linked D-galactopyranosyl residues, were relatively weak inhibitors. The combining sites of the lectin that reacts with pneumococcus type XIV polysaccharide appear, therefore, to be most complementary to 2-acetamido-2-deoxy-D-galactopyranosyl residues, probably in beta linkage.


Molecular immunological heterogeneity of the Salmonella zuerich [1, 9, 12, (46), 27] cell-wall polysaccharides.

Extraction of O specific polysaccharide from S. zuerich leads to three fractions (ZA, ZB, ZC). Polysaccharide ZB carries specificities 1, 27, and 46, present on the Salmonella cells. It exhibits a factor 27 that is very similar to that present on the S. typhi T2 1-minus 27-+ polysaccharide, a factor 1 that is close to that present on S. senftenberg polysaccharide, and a factor 46 that gives a very weak cross-reaction with anti-46 antibodies. Polysaccharides ZA and ZB are immunologically different and ZB contains two distinct fractions: ZB 1-minus devoid of Ofactor 1 and carrying the specificities 46 and 27 mostly, of not completely, on the same molecule (46, 27); and ZB1-+ carrying O factors 1, (46), 27. ZB 1-+ is composed of at least two different molecules: [1,(46)] precipitable with anti-1 antibodies but only coprecipitable with anti-46 antibodies; and (1, 27) precipitable with both anti-1 and anti-27 antibodies. Molecules [1, (46)] precipitate only part of the anti-1 antibodies precipitable by (1, 27). The smaller precipitation of anti-27 antibodies (when factor 27 is present together with factor 1 on the same molecule) and the coprecipitation, instead of precipitation, of anti-46 antibodies (when factors 46 and 1 are present on the same molecule) may be explained by a sterical hindrance between O-factors 1 and 27, and 1 and 46. The molecular, immunological heterogeneity of the polysaccharides extracted from S. zuerich would result from the presence on the cells of two kinds of O polysaccharides: one with, the other without O factor1, which is related to the presence of a side-chain of an alpha-D-glucosyl residue. A structure for S. zuerich polysaccharide is proposed.


The structure of capsular polysaccharide of the Pneumococcus type II.

The pneumococcus type II capsular polysaccharide (SII) is composed of singly-branched hexasaccharide repeating units, for which three alternative structures have been proposed. The correct structure has now been determined by consecutive eliminations of the sugar residues in the side chain. The terminal D-glucuronic acid group was eliminated by treating the fully methylated and esterified SIIpolysaccharide first with base, and then with weak acid. The hydroxyl group at C-6 in the penultimate D-glucose residue released by this elimination was transformed into the 6-deoxy-6-tosyl derivative, and the residue thereafter eliminated by treatment with base. As the side-chains were eliminated by these reactions, it is considered that they contain only two sugar residues, which thus excludes two of the three alternative structures. Structure 1 was further confirmed by subjecting SII to a Smith degradation, which yielded the tetrasaccharide L-Rhap-(1 yields 3)-L-Rhap-(1 yields 3)-L-Rhap-(1 yields 2-erythritol, characterised by methylation analysis.


In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes.

Exogenous E. coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes. No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP. Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP. The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP. Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP. Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed.


The effect of plasma on the reaction of cyclophosphamide with 4(-p-nitrobenzyl)-pyridine.

Cyclophosphamide is not structurally modified by blood plasma and may be recovered quantitatively from it. The apparent loss of alkylating ability of cyclophosphamide, as measured by its ability to react with 4-(p-nitrobenzyl)-pyridine (NBP), following treatement with plasma is not, as has been reported, due to metabolism of the drug but rather to an inhibition of the colorimetric reaction by a constituent of the plasma. This inhibition is strongly pH dependent, reaching 100% when the pH of the solution of cyclophosphamide in plasma is above 8, and falling to less than 20% when the pH is below 6, but extraction with chloroform at pH 7 separates cyclophosphamide from the inhibitor. Although the nature of the inhibitor has not been elucidated, its presence in plasma is of great importance in the quantitative determination of cyclophosphamide, and may also be of significance in the biological effects of cyclophosphamide and other alkylating agents in vivo.


Infrared and Raman spectra of phosphatidyl-ethanolamine and related compounds.

Infrared and Raman spectra of phosphatidylethanolamine from Escherichia coli, L-alpha-glycero-phosphorylethanolamine and 0-phosphorylethanolamine were obtained. Most of the bands were assigned to each vibrational mode based on the N deuteration effect, comparison of the intensity in the infrared and Raman spectra and the depolarization degree measurement in the Raman spectra. The spectra of phosphatidylethanolamine can be interpreted by assuming that the molecule takes the dipolar ionic structure both in non-polar solvent and in solid.


A spectrophotometric scanning technique for the rapid determination of plasma hemoglobin.

1. A spectrophotometric scanning technique was used to quantitate hemoglobin concentrations in cardiac surgery patients and patients with other hemolytic disorders. 2. The fractional absorption of a portion of the 577 nm band of oxyhemoglobin was used in conjunction was used in conjunction with the absorption coefficient of oxyhemoglobin to relate absorption to plasma hemoblogin concentration. 3. The spectrophotometric scanning technique offers a rapid and accurate means of assessing plasma hemoglobin concentrations even in the presence of usual interferences by bilirubin, turbidity and methemalbumin.


Enzymatic study of GM-1 gangliosidosis.

Two types of GM-1 gangliosidosis were studied biochemically. Type 1 liver accumulated non-lipid hexosamine in addition to GM-1 ganglioside, but there was no increase of hexosamine and GM-1 in type 2 liver. The optimum pH of liver beta-galactosidase of type 1 and type 2 was 5--6 while that of the normal control was 4.5. Type 1 brain beta-galactosidase showed a slightly acidic optimum pH, i.e. 4.0 in comparison with that of the normal control. The optimum pH of type 2 brain beta-galactosidase was 5.5, like the liver enzyme. The thermostability of liver beta-galactosidase was the same in type 1 and type 2, while that of brain was different. Beta-galactosidase of type 1 and type 2 liver is more stable at 42 degrees C than the normal control, but a different thermostability was observed in type 1 and type 2 brain. Liver beta-galactosidase of type 1 showed one peak each at acid and neutral pH, and type 2 liver had only one peak at neutral pH.


[Use of the measurement of gamma-glutamyltranspeptidase activity in serum to determine the success of cures for alcohol detoxification (author's transl)].

44 patients were studied during a 2-year period following a cure for alcohol detoxification. 29 patients (group A) did not start drinking again while 15 relapsed less than one year after their cure (group R). The average gamma GT activity (m) in mU per ml serum was, in group A, 148 (standard deviation, S.D. 184) at the beginning of the cure, 21 (S.D. 14) after one year and 19 (S.D. 13) after 2 years. During the same period there was no significant decrease in group R (cure: m 140, S.D. 161: 1 year: m 166, S.D. 164; 2 years: m 162, S.D. 163). On the other hand, all the subjects of group A who had a high gamma GT activity (greater than 29 mU/ml) at the start of the cure showed a decrease after one year.


Comparative investigations of some enzymatic parameters and liver scanning in the early detection of the malignant liver process.

In 30 patients with histologically verified malignant liver processes, the authors have examined the value of the serum activity of liver lactate dehydrogenase (LDH), alkaline phosphatase and gamma-glutamyltranspeptidase and in a number of patients, 5'-nucleotidase. These values were compared with the findings received from scintigraphy of the liver. Based on the results obtained, the authors have concluded that the activities of the gamma-glutamyltranspeptidase (gammaGT), 5'-nucleotidase and liver lactate dehydrogenase are more significantly increased in malignant processes of the liver than scintigraphy of the liver could register.


An automated technique for the analysis of plasma guanidino acids, and some findings in chronic renal disease.

A sensitive method for the quantitative determination of all guanidino compounds in human serum, including basic compounds such as methylguanidine, was developed by the adaptation of the Technicon automatic amino acid analyzer. Elution buffers with very high pH and molarity are employed for the elution of very basic compounds from the column. Automatic quantitative determination of the plasma guanidino compounds was performed with biacetyl alpha-naphthol, a specific reagent for the guanidino group. This method permitted the quantitative automatic determination of guanidino compounds in less than 1 ml of serum of normal or uremic patients. Guanidino propionic acid and guanidino succinic acid were identified in high concentrations in uremic sera. Methyl guanidine, and guanidine were in smaller concentrations. Guanidino acetic acid and guanidino butyric acid were present in normal plasma in quantities not significantly different from those in uremic plasma.


[Decrease in serum gamma-glutamyltranspeptidase following abstention from alcohol in cirrhotics (author transl)].

Gamma-Glutamyltranspeptidase activity of patients having an ascitic cirrhosis due to ethanol consumption is high (139 mU/ml) when the patient is still drinking at the time of the assay; it is lower when the patient had stopped drinking at least two months before the assay (49 mU/ml). On the other hand, in 10 patients out of the 11 who submitted to a second assay gamma-glutamyltranspeptidase decreases as soon as the patient abstains from alcohol. In 4 abstinent patients re-examined one year after the first measurement, the gamma-glutamyltranspeptidase activity had decreased to the reference values of Szasz. The half time of the return to normal has been estimated by extrapolation from the ethylic model at between 11 and 54 days. We conclude that the hyper gamma-glutamyltranspeptidase in cirrhotics is due to the ethanol impregnation and that repeated assays of the enzyme show whether the patient abstains from alcohol or not.


Fluoride determination in plasma by ion selective electrodes: a simplified method for the clinical laboratory.

A potentiometric method for the determination of fluoride (F-) in serum and plasma is proposed; it is based on a combination of the single-known-addition method and the electrode slope-by-dilution method. This procedure provides reliable results in extremely low measuring ranges down to 2.5 mug/l, where the electrode slope deviates markedly from Nernstian behaviour. In this method no electrode calibration is required and only one standard is necessary. 1 ml of plasma is sufficient for one analysis. Apart from a 5% enrichment of all samples with a concentrated total ionic strength adjustment buffer, no further preparation of the sample is required. The simplicity of the various pipetting and analytical steps, and also of the evaluation of the readings, may render this method highly suitable for the clinical laboratory. Investigations into the accuracy and precision of the method produced satisfactory results. The recovery in plasma amounted to 99.7%, even in the low measuring ranges. The discrimination capacity of the method amounts to 0.1 mug/l. With the apparatus and experimental procedures described, 18 plasma analyses per day can be performed even at low F- concentrations with which longer electrode stabilization periods are required. Storage of the plasma samples frozen at --20 degrees C for up to 8 weeks exerts no effect on the F- concentration. Problems of sample contamination and other disturbances are discussed. Determinations of normal values in 20 test subjects resulted in a mean value of 10.4 plus or minus 4.01 mug/l (Mean plus or minus S.D.). The modal value amounted to 9.5 mug/l, and the range was between 5.9 and 18.8 mug/l. The F- content of the drinking water supplied to this group of persons amounted to 180 mug/l. The importance of the method has been illustrated using a clinico-nephrological study as an example.


Separation of human alpha-amylase isozymes by electro-focusing and their immunological properties.

Human alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) was separated by electrofocusing. The amylases in serum and urine were seperated into two major isozymes with isoelectric points of pH 6.4-6.5 and pH 6.9-7.0, respectively, and one minor isozyme with an isoelectric point of pH 5.9. The amylases in saliva were separated into one major isozyme with an isoelectric point of pH 6.5 and two minor isozymes with isoelectric points of pH 6.0 and 6.9. The amylases in pancreatic juice gave one major peak at pH 7.0 and two minor peaks at pH 6.0 and pH 6.5. Repeated electrofocusing of each isozyme have only a single peak with a constant isoelectric point. The antigenicites of these three isozymes were also investigated using antisera to rat pancreatic and parotid amylases.


Linkage investigation of a large family with Reifenstein's syndrome.

Linkage analysis of a 116-member family with 11 males affected by Reifenstein's syndrome is reported. One X-chromosomal and eight autosomal markers were used. Close linkage can be excluded for P, K, MNS and Xg-a. A possibility of close linkage to ABO is discussed.


Comparison of the hypnotic activity of triazolam, flurazepam hydrochloride, and placebo.

Triazolam, 0.4 and 0.8 mg, flurazepam, 15 and 30 mg, and placebo were compared in a double-blind, randomized 5-night crossover study in 25 inpatient insomniacs. These patients all complained difficulty falling asleep; all said they usually slept less than 5 hr a nigh and woke up too early in the morning. Results of the patients' global evaluation of the medications shows that all of the treatments were rated significantly higher than placebo, with the exception of triazolam, 0.4 mg, which was not significantly different from flurazepam, 15 or 30 mg, or from placebo. In subjective evaluation of sleep onset, only triazolam, 0.4 and 0.8 mg, was rated faster than placebo. All 4 active medications increased duration of sleep. Triazolam, 0.8 mg, and flurazepam, 30 mg, were rated as providing deeper sleep than placebo while all treatments except flurazepam, 15 mg, decreased the number of awakenings below that on placebo. A significant dose-response curve was obtained with triazolam and flurazepam for some of the parameters. Very few adverse effects were reported. One patient reported feeling groggy and drowsy on 0.4 mg triazolam while 2 reported nightmares on placebo.


Effects of intravenous and oral propantheline and metoclopramide on ethanol absorption.

The separate effects of propantheline, atropine, and metoclopramide on ethanol absorption have been studied in man. Intravenous propantheline lowered blood ethanol levels after ingestion of a standard ethanol load. Oral propantheline, at dose levels currently recommended for therapeutic use, was without significant effect on ethanol tolerance, whereas the tolerance was reduced by oral atropine. Propantheline bromide tablets have been shown to undergo significant hydrolysis at alkaline pH in vitro. Metoclopramide, given intravenously and orally, significant elevated blood ethanol levels soon after ingestion of a standard ethanol load. It is suggested that when propantheline is selected as an anticholinergic for clinical use, there is need for greater awareness of the marked reduction in bioavailablity that results when the drug is administered at conventional therapeutic dosage by the oral as opposed to the intravenous route.


Ammonia and urea transport by the excluded human colon.

1. Ammonia and urea transport across the colonic mucosa was studied by a perfusion technique in four subjects with colonic exclusion for chronic hepatic encephalopathy. 2. Reduction of luminal pH inhibited net and unidirectional transport of ammonia from lumen to plasma, but net absorption from high luminal concentrations persisted at low pH. 3. Neither addition of urea to the perfusate nor intravenous infusion of urea produced a consistent increase in the colonic excretion of ammonia when ammonia-free solutions were perfused. 4. In one subject intravenous infusion of (15N)-ammonium chloride produced rapid labelling of colonic effluent ammonia and within 60 min the specific enrichments of ammonia in effluent and in arterial plasma were approximately equal. 5. During perfusion of nitrogen-free solutions, only small amounts of urea appeared in the effluent, suggesing limited permeability of the colonic mucosa to urea. 6. These results are discussed in relation to the equilibration of ammonia across the colonic mucosa by both ionic and non-ionic diffusion. The lack of evidence of 'juxtamucosal' (as opposed to luminal) ureolysis is in contrast to other observations on the intact colon. The possible reasons for and implications of this discrepancy are discussed.


Effect of bile-duct ligation on organelle marker enzymes in the liver and serum of rats.

1. Marker enzymes for the principal subcellular organelles of rat liver were asayed in the liver of rats 1 day and 8 days after bile-duct ligation or after laparotomy as a control procedure. 2. The microsomal enzymes in liver tissue showed complex changes. Benz[alpha]pyrene hydroxylase activity, predominantly found in the smooth endoplasmic reticulum, was decreased. Glucose 6-phosphatase activity and ribonucleic acid, which are localized predominantly in the rough endoplasmic reticulum, were increased. 3. The plasma membrane enzyme, alkaline phosphatase, increased in activity after bile-duct ligation. 4. No changes in mitochondrial enzyme activities were noted after 1 day but there was a 50% reduction 8 days after ligation. Lysosomal enzyme activities did not change in the liver tissue. 5. Liver catalase and D-amino acid oxidase activities showed a slight increase at 1 day postligation but a significant fall by 8 days. 6. Lactate dehydrogenase, a cytosol enzyme, showed a decrease in activity after 1 day but an increase in tissue activities 8 days after ligation. 7. Serum activities of mitochondrial, plasma membrane, microsomal, lysosomal and cytosol marker enzymes tended to increase post-ligation, particularly at 8 days. 8. Monoamine oxidase, a predominantly mitochondrial enzyme, was greatly elevated in the serum after 1 day but had returned to normal activities by 8 days.


Intestinal obstruction in the horse. Physical signs and blood chemistry.

Physical signs and blood changes were studied in horses with artificially produced obstructions of the duodenum and the small colon and simulated volvulus of the ileum. Horses with obstruction of the duodenum had the most violent physical signs and the shortest survival time. Blood changes were an initial rise in pH followed by acidosis, hyperkalemia and a decrease in HCO3 minus, Na+ and C1 minus. Obstruction of the small colon resulted in mild physical signs. The blood parameters recorded were normal. Simulated volvulus resulted in continuous colic. Blood changes were acidosis and hyperkalemia with a continuous decrease in HCO3 minus and C1 minus levels.


Pathogenesis of lead shot poisoning in the mallard duck.

Adult mallard ducks were administered steel pellets to determine the rate of excretion from the gastrointestinal tract. In separate studies the ducks were administered 5 number 6 lead pellets. Birds were examined for clinical signs and sacrificed at given intervals over a 20 day period to assess changes in tissue structure and concentrations of lead with time. The above studies were conducted in 2 groups of ducks, fed a low or a high fiber diet. The rate of steel pellet excretion on birds on the low fiber diet decreased with an increase in pellet size. Pellet excretion was greatly reduced in birds fed the high fiber diet. Administration of lead shot resulted in the development of green diarrhea, anorexia and weakness. It also produced high concentrations of lead in the blood, kidney, liver and bone with lower concentrations in skeletal muscle. The major lesions were destruction of the mitotically active proventricular epithelium and medullary osteocytes, destruction of pectoral muscle cells and the presence of intranuclear inclusion bodies in the proximal tubular epithelium of the kidneys. Birds on the high fiber diet demonstrated more severe clinical signs and higher concentrations of lead in the tissues.


Respirator weaning of the newborn: some practical considerations.

Weaning of neonates from assisted ventilation or continous positive pressure breathing is a complex process. Unfortunately, at present we lack sensitive indices to measure the infant's ability to sustain spontaneous breathing. In the absence of such criteria we should rely heavily on clinical findings correlated with available biochemical data. Good clinical acumen and experience is necessary for successful weaning.


Metabolism of dihalomethanes to carbon monoxide. II. In vitro studies.

Dibromomethane was metabolized to carbon monoxide and inorganic bromide by a rat liver microsomal fraction requiring both NADPH and molecular oxygen. This biotransformation was characterized with respect to time course, microsomal protein concentration, pH, and temperature. The metabolism of dihalomethanes to carbon monoxide followed the halide order; thus, diiodomethane yielded the greatest amount of carbon monoxide, whereas dichloromethane yielded the smallest amount. A KM of approximately 16 mM was established for dibromomethane while the Vmax was found to be about 8 nmol of CO per mg of microsomal protein per min. Cytochrome P-450 was found to bind dibromomethane to produce a type I binding spectrum. Pretreatment with phenobarbital increased both microsomal cytochrome P-450 levels and the rate of conversion of dibromomethane to carbon monoxide. Pretreatment with cobaltous chloride or storage of microsomal preparations at 4 degrees C resulted in parallel reductions of both cytochrome P-450 levels and the rate of formation of carbon monoxide from dibromomethane. SKF 525-A, ethylmorphine, and hexobarbital inhibited the conversion of dibromomethane to carbon monoxide. Microsomal preparations from rat lung metabolized this substrate at about 18% of the rate found in liver microsomes. The requirement for both NADPH and molecular oxygen, the inhibitory effects of SKF 525-A, ethylmorphine, and hexobarbital on the production of carbon monoxide from dibromomethane, and the correlation established between microsomal cytochrome P-450 levels and the rate of metabolism of dihalomethanes to carbon monoxide after treatments that alter the cytochrome to P-450 content suggest that these compounds are metabolized to carbon monoxide via a cytochrome P-450-dependent system.


Metabolism of R-(+)-and S-(minus)-pentobarbital by hepatic microsomes from male rats.

Hepatic microsomes from male rats hydroxylate R-(+)- and (-)-[2-14-C]pentobarbital to the diasterioisomeric 3'-alcohols: metabolite I (1'RS, 3'SR)- and metabolite II (1'RS, 3'RS)-5-ethyl-5-(3'-hydroxy-1'methylbutyl) barbituric acids. The rate is linear up to 1.5 mg of microsomal protein per ml of incubation mixture and for up to 16 min. The metabolism of R-(+)-pentobarbital led to production of nearly equal quantities of metabolites I and II with a total hydroxylation of 1.08 nmol/min/mg of protein. On the other hand, the S-(-) pentobarbital gave 3- to 5-fold less of metabolite I than II and a total hydroxylation of 1.42 nmol/min/mg of protein. The KM values for formation of metabolite I were 381 muM from R-(+)- and 241 muM from the S-(-)-pentobarbital, while for metabolite II they were 115 muM from R-(+)- and 68 muM from S-(-)-pentobarbital. These data suggest that there are at least two enzymes catalyzing the hydroxylation of pentobarbital, one to give metabolite II and another or others to give metabolite I.


The metabolism and disposition of aniline in the isolated blood-perfused liver of the rat.

In the isolated, blood-perfused liver of the rat, aniline is removed from the perfusion medium rapidly over the first 60 min, but more slowly over the following 120 min. The main metabolite found in the perfusion medium is an acid-labile conjugate of aniline which after 3 hr accounts for 33% of the aniline added, whereas p-aminophenol conjugates account for a further 13%. No free p-aminophenol could be detected in the perfusion medium and only a small amount in the bile. Aniline conjugates form the major metabolites found in the bile. A kinetic analysis of the data is presented and a two-compartment model is postulated in which aniline is removed for excretion in the bile or for metabolism from the first compartment. SKF 525-A, 0.2 mM, completely blocks the formation of p-aminophenol and inhibits the formation of the acid-labile aniline conjugate by 62%. There is also an increase in the size of the second compartment.


Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components.

Lung microsomal cytochrome P-450 was solubilized and purified 2-fold. NADPH-cytochrome c reductase (EC 1.6.2.3) was solubilized and purified 4-6 fold by three methods with use of sonication and detergent digestion followed by either DEAE-cellulose chromatography or ammonium sulfate fractionation. Benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities were reconstituted when NADPH-cytochrome c reductase and cytochrome P-450 fractions were combined. Reductase fractions prepared by sodium cholate digestion of microsomes were highly active in supporting the hydroxylation activity in the reconstituted systems, whereas those prepared with sodium deoxycholate were not. About twice as much NADPH-cytochrome c reductase was required for saturation of the 7-ethoxycoumarin deethylase activity as for saturation of the benzphetamine N-demethylase activity. A heat-stable lipid fraction was necessary for maximum hydroxylation activity. NADH did not support benzphetamine N-demethylation in the reconstituted system or increase the rate of reaction when added with NADPH.


Biliary excretion of diphenylhydantoin in the rat. Time-course studies.

The role of biliary excretion in the dose-dependent elimination of diphenylhydantoin (DPH) was investigated in the rat. During 6 hr, following iv injection of 10 mg of (14-C)DPH per kg, 28% of the radioactivity was excreted in bile and following the higher dose of DPH reached a plateau and remained constant over a period of 6 hr, whereas, with the lower dose, the excretion was a first order process (half-life 165 plus or minus 34 (SE) min). When the major metabolite of DPH,5-(P-hydroxyphenyl)-5-phenylhydantoin (P-HPPH, 14-C-labeled), was administered 1 and 10 mg/kg iv), the rates of biliary excretion of total radioactivity were first order, with half-lives of 49 plus or minus 3 and 57 plus or minus 4 min, respectively. Therefore, the conjugation of p-HPPH and the transfer of the conjugate from liver cells into bile were not saturable with the two doses studied. The data are consistent with the assumption that the dose-dependent elimination of DPH is due to the saturable conversion of DPH to p-HPPH.


The uptake and distribution of 14-C-mescaline in different organs of developing rat.

Rats of 1,4,8,12,20, and 60 days postnatal age were injected ip with 14-C-mescaline (50 nCi/g). The levels of mescaline and its deaminated metabolite, 3,4,5-trimethoxyphenylacetic acid, were examined in the brain, liver, heart, spleen, lung, and kidney at 30, 60, 90, and 120 min. Mescaline was rapidly taken up by all the organs examined. In general, the organs of younger rats accumulated much larger amounts than those of adult animals. Brain concentrated the lowest amounts in comparison with other tissues. In the brain, the uptake was the highest in 1-day-old rats and decreased with age. The disappearance of mescaline in various organs was comparatively slower in younger animals than in 20-day or older rats. Rats immediately after birth and uptake was the highest in 1-day-old rats and decreased with age. The disappearance of mescaline in various organs was comparatively slower in younger animals than in 20-day or older rats. Rats immediately after birth and up to 20 days of age metabolized mescaline less efficiently than adults. From the data, it appears that the blood-brain barrier for mescaline develops gradually with age but is not completely impermeable in adults.


Epoxide metabolites of protriptyline in rat urine.

Two epoxide metabolites of 5-(3-methylaminopropyl)-5H-dibenzo[a,d]cycloheptene (protriptyline) were identified in the urine of rats given 14-C-labeled drug. They were characterized by mass spectrometry, nuclear magnetic resonance spectrometry, and chemical reactivity as 10,11-dihydro-10,11-epoxy-5(3-methylaminopropyl)-5H-dibenzo[a,d]cycloheptene (I) and 10,11-dihydro-10,11-epoxy-5(3-aminopropyl)-5H-dibenzo[a,d]cycloheptene (II). Over twice as much I as II was excreted and together the two metabolites accounted for approximately 40% of the urinary radioactivity.


Lorazepam: glucuronide formation in the cat.

The excretion and metabolism of lorazepam was studied in domestic cats. After oral administration of 14-C-lorazepam (1 mg/kg), the mean percent of the dose excreted in urine was 47.3 plus or minus 6.7% (SD) and in feces 54.0 plus or minus 6.1% (SD). The main urinary metabolite was lorazepam glucuronide; its mean excretion over the first 3 days amounted to 29% of the dose (66% of urinary radioactivity). When 20 mg of unlabeled drug per kg was given, about 40% of the dose was excreted into urine as lorazepam glucuronide within 6 days. Conculsive evidence for the glucuronide structure was obtained by chemical analysis and mass spectrometry of the lorazepam conjugate isolated from cat urine. The radioactivity in urine which was not attributable to several minor metabolites. In plasma, both lorazepam and lorazepam glucuronide were present. These findings indicate that the cat is capable of using glucuronidation as a major route of conjugation, contrary to the many reports that cats conjugate exogenous materials poorly with glucuronic acid.


Timolol: a preliminary report of its pharmacological properties and therapeutic efficacy in angina and hypertension.

Timolol (MK 950) is a new beta-adrenergic receptor blocking drug. It has little or no membrane stabilising ('quinidine-like') or partial agonist (intrinsic sympathomimetic) activity and thus resembles sotalol. On a weight for.weight basis, timolol is more potent than sotalol or propranolol. A 2.5 mg dose of timolol causes about the same reduction in resting heart rate as 20mg of propranolol. Results of placebo-controlled and of comparative trials with other beta-adrenergic receptor blocking drugs, have shown that timolol effectively lowers blood pressure without producing orthostatic or exercise hypotension. Findings of an international multicentre trial in angina pectoris, indicate that timolol is effective in reducing the frequency of anginal attacks and the consumption of glyceryl trinitrate for their relief.


Metabolism and disposition of N-(4-(5-nitro-2-furyl)-(2-14-C)thiazolyl)acetamide in the rat.

The metabolism of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), a carcinogen for the mouse, rat, dog, and hamster, was investigated in the rat. Radioactive NFTA administered ip to young and lactating female rats was rapidly absorbed through the peritoneum and deposited in the urine, intestinal contents, and feces. Less than 0.5% of the radioactivity was expired as CO2 during the first 24 hr. Two yellow metabolites, accounting for 5 and 27% of the radioactivity in the urine, were found by paper chromatography. One metabolite retained the 2-amino-4-(5-nitro-2-furyl)thiazole moiety. Less than 0.5% of the radioactivity in the urine was due to unchanged 14-C-NFTA. The radioactivity level in the visceral organs reached a maximum 2 hr after dosing and then gradually decreased. Liver contained considerably more radioactivity than did other visceral organs; this was mainly distributed in the cytosol and 900g pellets. Fifty per cent of the radioactivity in the liver was bound to the hot trichloroacetic acid (TCA)-insoluble fraction; that bound to the cold TCA-insoluble fraction increased gradually from 66.9% at 2 hr to 82.6% at 24 hr. The radioactivity bound to the cold and hot TCA-insoluble fractions in kidney remained at 30% and 20%, respectively. Nitroreduction of 14-C-NFTA by microsomes, as a prerequisite of the binding of metabolite to microsomal protein, was demonstrated. Addition of L-cysteine or reduced glutathione to the incubation mixture did not alter the nitroreductase activity of the microsomes; however, binding of radioactivity to protein was significantly decreased. Addition of other amino acids did not significantly decrease the nitroreductase activity or binding to protein. These results suggest that reduced NFTA binds to the protein sulfhydryl groups. Since 14-C-NFTA binds to macromolecules in vivo,nitroreductionmay be important for the metabolism of 5-nitrofurans.


Hydroxylation of 14-C-niflumic acid by phenobarbital-induced rat liver microsomes.

Hydroxylation of niflumic acid to 4'-hydroxyniflumic acid and 5-hydroxyniflumic acid by rat liver microsomes from untreated animals or phenobarbital-pretreated animals has been demonstrated. Pretreatment of rats with phenobarbital (50 mg/kg) increased the 4'-hydroxylase activity 53%, whreas the 5-hydroxylase activity was increased 205%. Niflumic acid inhibited of 4'- or 5-hydroxylase was observed with SKF 525-A. WIN 13099 inhibited both hydroxylases noncompetitvely. With freshly prepared microsomes, acetone, at concentrations frozen and aged microsomes, maximum enhancement of 4'-hydroxylase was obtained at 5% acetone; little or no effect on 5-hydroxylase was observed at the same concentration. At 10% acetone, the 4'- and 5-hydroxylases were inhibited 33 and 73% respectively. The data indicate that at least two different enzyme systems are involved in the hydroxylation of niflumic acid by the microsomes of rat liver.


Adrenal hormones and increase of liver tyrosine aminotransferase and tryptophan pyrrolase activity after immobilization in rats.

In adrenomedullectomized rats the postimmobilization increase of liver tyrosine aminotransferase and tryptophan pyrrolase activity was similar as in intact animals, wherease in adrenalectomized rats this response was completely absent. In intact animals a positive correlation between the magnitude of the response of both enzymes and the duration of immobilization and/or the extent of plasma corticosterone increase was observedmit is concluded that the postimmobilization hyperactivity of both enzymes arises exclusively as a consequence of hypercorticosteronaemia, catecholamines and other hormones being without any influence on this response.


LH-RH interactions with pituitary receptors: Properties and characterization.

The plasma membrane initiator system for actions of LH-RH was characterized. Binding of 125-I-LH-RH to the anterior pituitary plasma membrane was studied at 4 degrees C and at 37 degrees C. Two binding sites were indentified which possessed apparent affinity constants of 2 times 10- minus 8M and 2 times 10- minus 7M, respectively. The high affinity binding site was competible and saturable by TRH. These data suggest that the low affinity binding site is the physiological receptor for mediation of LH-RH stimulated LHrelease. LH-RH-receptor interaction has been shown to be a temperature and pH-dependent process. Less binding was observed at 37 degrees C than at 4 degrees C. Below pH 6 and above pH 8 a sharp drop in LH-RH binding to the cell membrane was recorded. Ca++, Mg++ and Mn++ were shown to inhibit the binding. Mn++ (16 mM) showed 50 percent inhibition of binding of LH-RH to the anterior pituitary plasma membrane fractions. On the other hand, increasing ionic strength enhanced LH-RH binding. The results of our study on the LH-RH-receptor systems offer a new approach to the study of LH-RH actions and facilitate the interpretation of earlier observations.


Calcitonin inhibition of bone cell metabolism in vivo: an experimental study in dogs.

Calcitonin (CT) has been shown to act by decreasing the output of calcium from the bone, but the precise mechanism that brings about this change is still not fully understood. The present investigation was designed to study the effect of CT on the metablic activity of bone cells, utilizing changes in oxygen consumption and carbon dioxide production as indices of tissue metabolism. Arterio-venous gradients of calcium, inorganic phosphate, hydroxyproline, oxygen, carbon dioxide and hydrogen ions were studied across the canine femur. The results showed that the hypocalcemic action of CT was accompanied by a corresponding decrease in free hydroxyproline output from the bone, demonstrating that decreased bone resorption was responsible for the fall in plasma calcium levels. Reduced oxygen consumption and decreased carbon dioxide production by the bone showed that CT acted by inhibiting the metabolic activity of bone cells in vivo. It is speculated that CT may have acted by depressing mitochondrial respiration.


Effect of acute metabolic acidosis on vasopressin-dependent cyclic AMP in rat kidney.

Acidic media have been reported to inhibit the hydro-osmotic effect of vasopressin in toad bladders, probably through inhibition of the cyclic AMP system. However, the mechanism of inhibition of the cyclic AMP system is controversial. Therefore, that inhibitory mechanism was further investigated in rat kidneys. The antidiuretic response to vasopression was significantly inhibited in animals with metabolic acidosis. The inhibition of the antidiuretic response was associated with a smaller than normal increase of urinary excretion of cyclic AMP after the iv injection of vasopressin. In in vitro experiments, both the increase of cyclic AMP concentration in renal medullary slices and the activation of adenylate cyclase in medulla by vasopressin were significantly less in acidic than in control media. These findings suggest that medabolic acidosis inhibits the antidiuretic effect of vasopressin by inhibiting the vasopressin-dependent cyclic AMP system in the kidney. Acidic media also inhibited cyclic AMP-phosphodiesterase. These dual effects of acidosis on adenylate cyclase and cyclic AMP-phosphodiesterase may explain the conflicting findings observed in the experiments on toad bladders.


A 3'-deoxymononucleotide-producing nuclease from the marine sponge Verongia aerophoba. II. General properties.

The properties of a 3'-deoxymono-nucleotide-producing nuclease highly purified from the marine sponge Verongia aerophoba are described. The hydrolysis of heat-dentured DNA-proceeds at about five fold the rate of native, double-stranded DNA. The enzyme behaves as an exonuclease by producing 3'-deoxymononucleotides. Kinetic analysis by means of initial hyperchromicity at 260 nm indicates that the enzyme requries 120 mmol/l of mono-valent and 30 mmol/l of divalent cations with a clear dependence on ionic radius. The enzyme has  a pH optimum at 4.7, the pH value of the isoelectric point is 6.1. The molecular weight has been evaluated at 62,000.


Renal glutaminase in postnatal and adult rats.

Properties of renal phosphate-dependent glutaminase (EC 3.5.1.2), assayed in tissue homogenates, were compared in adult, 2-week-old, and newborn rats. Vmax, Km glutamine, pH optimum, inhibition by glutamate, activation by phosphate, intracellular distribution, and the possible presence of activators or inhibitors were examined. Although Vmax increased threefold during postnatal development, no major differences in the properties of the enzyme at the three stages of development were noted. It was concluded that the enzyme protein remains the same throughout development, both in biochemical properties and intracellular location, but that more of it is accumulated or is converted to an active state as the kidney matures.


The serum activity of glucose-6phosphatase and 5'-nucleotidase during human pregnancy.

An attempt has been made to show that the increase in enzyme activities in sera of pregnant women found with glucose-6-phosphate and adenosine 5'-monophosphate as substrates (described as glucose-6-phosphatase and 5'-nucleotidase) was due to the increase in alkaline phosphatase. The three enzyme activities has pH optima and heat stability characteristics of alkaline phosphatase. The response to the action of inhibitors and activators was typical for alkaline phosphatase. There was an identical increase in all three enzyme activities during pregnancy. As a control similar investigations were made with liver and placental tissue extracts.


Two forms of 4-aminobutyrate transaminase in guinea pig brain.

4-Aminobutyrate transaminase (GABA-T, 4-aminobutyrate alpha-oxoglutrate aminotransferase, EC 2.6.1.19) is an enzyme that inactivates the inhibitory neurotransmitter, GABA, but its pharmacological function is uncertain. Two forms of guiena pig brain GABA-T were isolated by DEAE-cellulose chromatography and designated as GABA-T-I and II, corresponding to an anionic and a cationic form. The enzymes were inhibited by high concentrations of a cationic form. The enzymes were inhibited by high concentrations of alpha-oxoglutrate (alpha-KG). Kinetic consists for GABA, when determined at pH 7.9 adn 1 mmol/l alpha-KG, were 0.74 mmol/l. GABA-T activity was inhibited by chloride and other anions. Kinetic analysis revealed chloride ion as a conpetitive inhibitor against GABA, but the Ki values differed among GABA-T-I and II (Ki equals 120 and 60 mmol/l, respectively). Similar  degrees of difference were observed with acetate and lactate ion. These results suggest that GABA-T-II may regulate the GABA level in the inhibitory neurons and may play a similar functional role as that exhibited by monoamine oxidase in other synapses.


Activities of tyrosine, alanine and aspartate aminotransferases of fetal rat liver in organ culture.

The levels of tyrosine, aspartate and alanine aminotransferases of fetal rat liver were measured and compared with values reported in the literature. Incubation of explants of fetal liver in organ culture resulted in spontaneous increases in tyrosine and alanine aminotransferases, and decrease in aspartate aminotransferase.


D-asparatate oxidase in the thyroid gland.

D-Aspartate oxidase was isolated from the pig thyroid gland and purified over 600 times. The enzyme was obtained in an inactive form of apoenzyme and was activated by FAD. It was specific towards the D-form of aspartic acid, had no effect on the L-form, and was also inactive towards other monocarboxlyic D-amino acids. The enzyme was only slightly active towards D-glutamate. The Michaelis constant based on the Lineaweaver-Burk plot was 5 mmol/l. The optimum pH was 8.7. D-Aspartate oxidase was inhibited by KCN in concentrations varying from 0.05 to 1 mmol/l. The biological role of this enzyme in the thyroid gland is discussed.


Activation energy, relative substrate specificity and optimum pH of guanase from humanrat, mouse and guinea pig sera and tissues.

Some properties of serum and tissue guanase from man, rat, mouse and guinea pig have been studied. Serum guanase differs from tissue guanase in activation energy in the human species and in guinea pig. The mouse and rat brain enzyme differs from the liver and kidney enzyme. The guanase of each species differs from the enzyme of the other species in one or more properties.


Effect of magnesium ion (Mg2+) and the magnesium adenosine triphosphate ion (MgATP2-) on pigeon liver pyruvate carboxylase.

Kinetic methods have been used to determine whether Mg2+ and MgATP2- play an important role in regulating pigeon liver pyruvate carboxylase [pyruvate: CO2 ligase (ADP), EC 6.4.1.1]. Mg2+ not only forms a complex with ATP4- (MgATP2-) but is also required for the enzyme activation (and probably for the binding of MgATP2- to this enzyme). Contrary to the results of other investigators, the MgATP2- complex was not found to activate pigeon liver pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP2-. Excess Mg2+ induced allosteric stimulation of the enzymatic activity at different concentrations of MgATP2-. With different Mg2+ concentrations, changes also occurred in the apparent Km, Vmax and Rs values. Without excess of Mg2+ (heterotropic effector) only about 2% of the total enzymic activity available could be demonstrated in the presence of MgATP2-. It is concluded that Mg2+ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg2+ may bind either to a specific effector site, at the active site, or at the binding site for MgATP2- which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate.


Flavin-nicotinamide biscoenzymes: models for the interaction between NADH (NADPH) and flavin in flavoenzymes. Reaction rates and physicochemical properties of intermediate species.

1. Flavin-nicotinamide biscoenzymes covalently linked by two, three or four methylene groups through positions N(10) of the flavin (Fl) and (N1) of the nicontinamide (Nic) form long-wavelength-absorbing, intramolecular complexes when the flavin part of the molecule is reduced specifically. The energy of the long-wavelength transition is minimal and its intensity maximal for (see journal for formula). 2. The increasing proximity of the positively charged nicotinamide lowers the pK-value of dihydroflavin deprotonation up to 1.7 units and the flavin oxidation-reduction potential becomes more positive up to 116 mV. 3. Specific reduction of the nicotinamide part of the biscoenzymes yields transient, long-wavelength-absorbing complexes. The energy of the long-wavelength transition is minimal and its intensity maximal for the complex (see journal for formula). 4. The rate of intramolecular flavin-dependent dihydronicotinamide dehydrogenation is highest for (see journal for formula), about 3 times slower for (see journal for formula) and 100 times slower for (see journal for formula). 5. The results obtained in this study are consistent with a reaction mechanism that involves formation of a charge transfer complex between reduced nicotinamide and oxidized flavin and rate-limiting heterolytic breakdown into products.


Purification and kinetic properties of chalcone-flavanone isomerase from soya bean.

The chalcone-flavanone isomerase from soya bean seed has been purified 8300-fold. A molecular weight of 15,600 plus or minus 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form. The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.


The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.