Metabolic activation of norethisterone (norethindrone) to an irreversibly protein-bound derivative by rat liver microsomes.

Norethisterone, specifically labeled with tritium, was incubated with hepatic microsomes of rats. About 2% of 3H radioactivity was irreversibly incorporated into the microsomal protein. This protein binding of norethisterone (about 0.7-1.6 nmol/mg of microsomal protein) was dependent on oxygen, NADPH, substrate concentration, and microsomal protein content and could be inhibited by carbon monoxide. Glutathione and other cysteine derivatives with free sulfhydryl groups diminished the microsomal protein binding diminished the microsomal protein binding as did the addition of bovine serum albumin. Norethisterone-derived radioactivity was also irreversibly bound to albumin. Solvent-extraction and charcoal-adsorption methods were employed to prove the irreversible nature of this binding. After trypsin digestion of albumin and microsomal protein loaded with norethisterone, peptides which were labeled with 3H could be isolated. To explain our results, a metabolic bioactivation of norethisterone to norethisterone-4,5-epoxide, catalyzed by the microsomal mixed-function oxidase cytochrome P-450, is proposed.


Metabolism of triamterene in the rat.

The metabolism of 14C-triamterene (TA), 2,4,7-triamino-6-phenylpteridine, was investigated in rats. After administration of 14C-TA (2 mg/kg, sc), 45% and 50% of the total radioactivity was excreted in urine and feces, respectively, during 72 hr. 14C-TA and its metabolites were separated by paper chromatography and countercurrent distribution. Unchanged TA in the urine and feces accounted for 72-79% of the dose. The major metabolites in the excreted dose were free p-hydroxytriamterene (10-15%) and its conjugate (1-5%). Three hours after administration of the drug, the major metabolites were not found in gastrointestinal contents or urine when the bile duct was cannulated. They were formed in the liver and secreted in bile. A minor unidentified metabolite (2% of the dose) was also formed in the liver and excreted in urine and feces.


Biotransformation of a 1,5-benzodiazepine, triflubazam, by man.

The biotransformation of triblubazam (ORF 8063; 1-methyl-5-phenyl-7-trifluoromethyl-1H-1,5-benzodiazepin-2,4-[3H,5H]-dione) was studied in man. Seven male subjects received a chronic regimen of orally administered triblubazam for 19 consecutive days and their urine was collected for 29 days. Seven urinary metabolites were isolated by application of Sephadex LH-20 column chromatography and preparative thin-layer chromatography. Six of the purified compounds were subsequently characterized by utilizing thin-layer and gas-liquid chromatography and infrared, nuclear magnetic resonance, and mass spectrometry. The structure of a seventh metabolite was established by the use of an enzymatic assay involving catechol O-methyl-transferase. These compounds included unchanged triflubazam, the N-desmethyl catechol derivative, the 4'-hydroxyphenyl derivative, N-desmethyltriflubazam, the N-desmethyl dihydrodiol derivative, the N-desmethyl-4'-hydroxy compound, and the N-desmethyl-3'-methoxy-4'-hydroxy derivative. Unlike the situation in the metabolism of 1,4-benzodiazepines by man, no C3-hydroxylated derivatives of triflubazam were isolated. The metabolism of triblubazam by man is characterized by extensive N-demethylation, aromatic hydroxylation, aromatic O-methylation, and dihydrodiol formation.


Physiological disposition and metabolism of timolol in man and laboratory animals.

Timolol [3-(3-tert.-butylamino-2-hydroxypropoxy)-4-morpholino-1,2,5-thiadiazole], was rapidly absorbed, metabolized, and effectively excreted in man, rats, and dogs. Peak plasma levels of timolol-14C were observed in these species 1-2 hr after oral administration. Generally, less than 20% of the radioactivity was present in the plasma in the unmetabolized form. The intact drug had a plasma half-life of 28 min in the rat, 48 min in the dog, and 5.5 hr in man. After oral administration of timolol-14C to humans approximately 72% of the dose was excreted in 84 hr, with 66% in the urine and 6% in the feces. In the rat, 58% of an oral dose was excreted in the urine and 26% in the feces. The dog excreted 68% of an oral dose in the urine and 19% in feces in 72 hr. Following intravenous administration, rats excreted 50% in the urine and 28% in the feces, which suggests that extensive biliary excretion occurred. Timolol was extensively metabolized. Approximately 50% of the radioactivity was identified in dog urine as the lactic acid metabolite. An additional metabolite was tentatively identified as the 3-oxomorpholino derivative of timolol. Approximately 20% of the dose in man was excreted in the urine unchanged. Two metabolites, resulting from cleavage of the morpholine ring, were identified as 1-tert-butylamino-3-[4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-xloxyl-2-propanol, accounting for 10% of the urine radioactivity, and t-tert-butylamino-[4-(N-2-hydroxyethylglycolamido)-1,2,5-thiadiazol-3-yloxy]-2-propanol, accounting for 30%. A minor metabolite, resulting from hydroxylation of a terminal methyl group, accounted for an additional 3% of the urine radioactivity.


Effect of chlorination on the distribution and excretion of polychlorinated biphenyls.

The distribution and excretion of four 14C-labeled polychlorinated biphenyls (PCB), 4-chloro-, 4,4'-dichloro-, 2,4,5,2',5'-pentachloro-, and 2,4,5,2',4',5'-hexachlorobiphenyl, were studied in the rat. Total radioactivity was determined in the major organs and tissues at times varying from 15 min up to 42 days after an intravenous injection of a single 0.6-mg dose of each of the PCB's per kg. Each of the PCB's was removed rapidly from the blood and initially stored largely in the liver and muscle. The rates of the subsequent redistribution of the PCB's and their metabolites to the adipose tissue and skin and/or excretion could be related to the degree of chlorination. The relative amounts of each PCB and its metabolites in each major tissue were determined at selected time points. The percentage of the total radioactivity accounted for by metabolites of the PCB's decreased as the chlorination of the PCB's increased, but storage of metabolites did not account for a significant portion of any of the PCB doses. Excretion accounted for more than 90% of the dose of the mono-, di-, and pentachlorobiphenyls during the time period studies, whereas extrapolation of the hexachlorobiphenyl data indicated that less than 20% of the dose would ever be excreted. The rates of elimination of the PCB's were apparently determined primarily by their rates of metabolism. The effect of degree and position of chlorination on the rate of metabolism of the biphenyl molecule is discussed.


Species differences in benzene hydroxylation to phenol by pulmonary and hepatic microsomes.

The metabolism of benzene to phenol by microsomal preparations from lung and liver has been compared in hamsters, rats, and rabbits. There were wide differences in the apparent Vmax of benzene hydroxylation among the various species and tissues and smaller differences in the apparent KM values for benzene hydroxylase. Benzene can inhibit its own metabolism in vitro when present in high concentrations. Phenol was the only metabolite of benzene identified, under the conditions of the assay, in incubation mixtures containing microsomes from lung or liver of any of the three animal species. When incubated with microsomes under the conditions used to measure benzene metabolism, phenol was further metabolized in liver but not in lung preparations. Phenol metabolism was almost completely inhibited when 11.2 mM benzene was included in the incubation mixture containing hepatic microsomes. The variation in rates of benzene hydroxylation by microsomal preparations from lungs or livers of the three animal species was similar to the variation in rates of benzypyrene hydroxylation in the same preparations.


A study of the dynamics of imipramine accumulation in the isolated perfused rabbit lung.

An isolated perfused rabbit lung preparation (IPL) was used to study the uptake, accumulation, and efflux of the tricyclic amine imipramine (IMIP). The rate of IMIP uptake into the IPL was resolved into two exponential components (rates 1 and 2 of uptake). The initial velocities for these uptake components were linearly related to the concentration of IMIP in the perfusate (Cp). This linear relationship indicates that IMIP accumulated in the IPL by diffusion and/or binding. The steady-state accumulation of IMIP was obtained by integration of the exponential expression relating the rate of IMIP uptake to time. The amount of IMIP accumulated at steady state by rate 1 was linearly related to Cp, whereas the amount of IMIP accumulated at steady state by rate 2 was saturable with respect to Cp. These steady-state data are in agreement with the steady-state accumulation data previously reported from experiments with the recirculating blood-perfused rabbit lung. In the absence of IMIP in the perfusate. IMIP that had previously accumulated in the IPL effluxed from the lung at three rates (t1/2 = 18 sec, 58 sec, and 8.25 min), which indicates that accumulated IMIP was in at least three pools in the lung. In addition, a noneffluxable pool was detected which was not the result of irreversible binding to tissue.ated in pools 1 and 2 by rate 1 of uptake. The IMIP in pool 3 and in the noneffluxable pool was accumulated in the IPL by rate 2 of uptake. Efflux pool 3 and the IMIP accumulated at a steady state by rate 2 of uptake were resolved by a Hofstee plot into a biphasic curve indicating two types of binding sites. The noneffluxable pool of IMIP in the IPL was saturable with increasing Cp and represented approximately 30% of the IMIP accumulated by rate 2 into pool 3. Rate 2 of uptake, pool 3, and particularly the noneffluxable pool of IMIP in the IPL are possibly responsible for the accumulation and persistence of Imip in the lung as seen in whole body distribution studies. The physicochemical properties of IMIP and other compounds known to be accumulated in lung tissue are discussed in relation to the possible involvement of lung phospholipids and the compartmentalization of IMIP in the concentric lamellar organelles of the lung.


Biliary excretion products of 1-[1-14C]naphthyl-N-methylcarbamate (carbaryl) in rainbow trout (Salmo gairdneri).

Uptake and metabolism of 1-naphthyl-N-methylcarbamate (carbaryl) were studied in rainbow trout (Salmo gairdneri) exposed to 14C-carbaryl in water. A high degree of biliary concentration was indicated by a 24-hr bile to water 14C ratio of nearly 1000:1. Four 14C-labeled compounds were purified from bile and two have been identified with reasonable certainty as unchanged carbaryl and 1-naphthol glucuronide. 5,6-Dihydro-5,6-dihydroxy-1-naphthyl-N-methylcarbamate was tentatively identified by thin-layer chromatography. The results indicate that carbaryl can be metabolized in rainbow trout via pathways similar to those reported in mammals and that bile analysis may be helpful in monitoring fish for exposure to this compound.


Effect of 3-methylcholanthrene pretreatment on glucuronidation and sulfation in perfused rat liver.

The metabolism of p-nitro[14C]phenol (PNP) was studied in perfused rat liver. Metabolites were identified in perfusate and bile. The perfusion system containing 0.5 mM PNP, at the start, converted the phenol within 2 hr to p-nitrophenyl glucuronide (PNPGA), 31.4% in perfusate and 21% in bile; p-nitrophenyl sulfate (PNPS), 22% in perfusate; and p-nitrophenyl glucoside (PNPG), between 2 and 4% perfusate. Biliary excretion of PNPS and PNPG accounted for less than 2% of PNP. An apparent maximal rate of PNPGA synthesis by the perfused liver, estimated from the rates of appearance of PNPGA in perfusate and bile, corresponded well to the glucuronyl-transferase activities determined in "native" liver homogenates. Sulfation of PNP in the perfused liver exhibited two apparent maximal rates of synthesis, as determined from the rates of appearance of metabolite in the perfusate. A slower rate (R1) occurred when PNP concentration was between 0.13 and 0.5 mM, and a faster rate (R2) when PNP was approximately between 0.025 and 0.13 mM in the perfusate. Possible significance of these findings is discussed. Pretreatment of rats with 3-methylcholanthrene (3MC) increased the apparent maximal rate of PNPGA production by the perfused liver by a factor of 1.7, and biliary excretion of PNPGA by a factor of 1.5. The latter increase was attributed to the increased rate of synthesis of the glucuronide rather than to an increase in the biliary transport maximum, since the 3MC treatment produced no significant effect on the biliary excretion of performed PNPGA added to the perfusate. The 3MC pretreatment increased 1.5-fold the rate of sulfation of PNP, as judged by the increase in R2.


Effect of beta and beta2 adrenoreceptor stimulants infused intrapancreatically on glucagon and insulin secretion.

L-Isoproterenol was infused at a dose of 20 pmol/kg/min for 10 min into the cranial pancreaticoduodenal artery in anesthetized dogs. Arterial plasma glucose, blood flow, and plasma concentrations of both glucagon and insulin in the cranial pancreaticoduodenal vein were significantly enhanced during the infusion, resulting in a greater increase of bihormonal output. Intrapancreatic pretreatment with propranolol abolished all of the isoproterenol-induced increases except for glucagon secretion which was suppressed only in part. Pretreatment with practolol, a specific receptor blocker of the beta1 type, did not exert any discernible inhibiting effect upon the isoproterenol-induced enhancement. Intrapancreatic infusion of trimetoquinol, a selective receptor stimulant of the beta2 type in some mammals, at an equimolar dose caused similar increases in plasma glucose, pancreatic venous blood flow, and bihormonal output when compared to those induced by isoproterenol. Pretreatment with a larger dose of propranolol totally abolished the trimetoquinol-induced enhancement of both glucagon and insulin secretion. Pretreatment with an isomolar dose of practolol, in contrast, did not show any suppressive effect on the parameters investigated. There was a dose-dependency in the bihormonal responses to trimetoquinol. Another beta2 receptor agonist, salbutamol, also significantly raised plasma glucose, pancreatic venous blood flothough to a lesser extent than did trimetoquinol. These results indicate that the adrenergic control over the function of the endocrine pancreas through beta adrenoreceptors may be mediated mainly via those of the beta2 type.


Sex hormones and tyrosine hydroxylase activity in vascular and adrenal tissue.

Vascular tyrosine hydroxylase (TH) activity did not appear to be affected by the sex hormones. There were no differences in enzyme activity in the mesenteric artery or vein taken from male and female normotensive or spontaneously hypertensive rats. Castration of either male or female rats did not alter mesenteric artery or vein TH activity, and the administration of estradiol, progesterone, or testosterone also had no effect on vascular TH activity. However, the sex hormones did alter activity in other tissues. Estradiol and progesterone administration to intact female rats increased adrenal TH activity, whereas castration of the male rat decreased it. Although the sex hormones were not important regulators of TH in blood vessels, vascular TH activity did appear to be under some hormonal regulation since hypophysectomy decreased mesenteric artery enzyme activity. Hypophysectomy studies also indicated that adrenal TH activity was under some hormonal regulation.


Aerobic photodegradation of X(N) chelates of (ethylenedinitrilo)tetraacetic acid (EDTA): implications for natural waters.

The behavior of several metal chelates [X(N)] of 1-14C-(ethylenedinitrilo) tetraacetic acid [EDTA] under irradiation from a wide spectrum Xenon arc lamp has been studied. In static tests at pH 4.5, chelates of Mn(II), Fe(II) and Co(II) photodegraded to give 14CO2 and CH2O. Chelates of Na(I), Mg(II), Ca(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) did not exhibit any significant photodegradation as measured by 14CO2 evolution. It is concluded that the photodegradation of Fe(III)-EDTA by sunlight will prevent EDTA build up in natural waters.


Adsorpition of tryptophan onto calcum carbonate surface.

Adsorption of tryptophan onto CaCO3 at constant ionic strength (0.05 M Nacl) and from dilute aqueous solution (10(-4) M to 10(-3) M tryptophan) is reported. Adsorption was primarily determined by the charge characteristics of both the adsorbate amino acid and adsorbent CaCO3. When both adsorbate and adsorbent are semicharged, tryptophan ions are expelled away from the CaCO3-solution interface. Tryptophan is only removed by CaCO3 in a narrow pH range, 6.0 greater than pH less than 8.5 within which CaCO3 has positive charges, and tryptophan is negatively charged. The pH of zero point of charge, pHzpc, of CaCO3 was also determined by alkalimetric tritration and coagulation techniques and a value of 9.50 +/- 0.5 was found. These preliminary finding demonstrate primarily that interfacial chemical reactions play an important role in the temporal and spatial transformation of dissolved organic matter in natural water systems.


Ion-binding to phospholipids. Interaction of calcium and lanthanide ions with phosphatidylcholine (lecithin).

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations greater than 0.1 mM. 1H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1--10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+ -lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA approximately 30 l/mol was calculated at lanthanide concentrations greater than 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cation-lecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.


Highly purified glutamine transaminase from rat brain. Physical and kinetic properties.

Glutamine transaminase from rat brain was purified to a high degree. The isolated enzyme appeared to be homogeneous by electrophoresis on polyacrylamide gel. The molecular weight was found to be approximately 98 000; the enzyme is probably composed of two subunits. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents are strong indications for the presence of pyridoxal phosphate. The enzyme showed maximal activity at pH 9.0 to 9.2. Of the amino acids tested, none could replace glutamine in the transamination reaction. Glyoxylate and phenylpyruvate was found to be the best amino acceptors. The Km values for glutamine and glyoxylate were 0.6 and 1.5 mM, respectively.


Influence of various factors on the recognition specificity of tRNAs by yeast valyl-tRNA synthetase.

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl-tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non-cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions. The influence of various factors on the discrimination by the enzyme between the cognate and the non-cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constant either in the cognate or in the non-cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific of non-specific interactions must be identical. On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.


A fluorescence study of the temperature-dependent polymerization of bovine beta-casein A1.

The intrinsic fluorescence properties of bovine beta-casein A1 solutions have been studied under a variety of conditions. The protein shows both tyrosine and tryptophan fluorescence emission, the former being more pronounced at low temperature (5 degrees C) and in the presence of urea. Approximate relative quantum yields for the tyrosine and tryptophan residues were determined using free tyrosine and free tryptophan respectively as standards. The tryptophan emission intensity of beta-casein shows an increase with temperature indicating a temperature-dependent transition of the protein. Two-state analysis of the emission-intensity--temperature data yielded positive enthalpy and entropy values for the transition over a range of protein concentrations (0.018--0.18%). A marked protein concentration effect was apparent which indicated that the transition was a consequence of polymerization rather than a prerequisite for polymerization. The size of the polymers appeared to be concentration-dependent. Decreased ionic strength and increased pH both caused a reduction in the increase in emission intensity when the temperature was increased. At higher pH both the enthalpy and the entropy for the transition were reduced. Experimental precision was insufficient to allow the effect of decreased ionic strength on these parameters to be determined. Many of the properties of the temperature-dependent transition can be explained if the transition is considered to be one of micelle formation.


The location and function of cytochrome c2 in Rhodopseudomonas capsulate membranes.

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate. Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9-aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift. It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophors and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.


Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.


Lysosomal enzymes as agents of turnover of soluble cytoplasmic proteins.

The degradation of cytosol proteins in vitro by purified cathepsin D and cathepsin B1 and by mixtures of lysosomal enzymes, was studied. By means of a double-labelling method, it was shown  that the relative rates of degradation of cytosol proteins by the purified enzymes and by mixtures of enzymes under a wide range of conditions in vitro correlated well with their relative rates of turnover in vivo. The complex mixture of cytosol proteins was degraded less rapidly after denaturation than in the native state, both by the purified proteases and by the mixture of lysosomal enzymes. This contrasts with previous results on proteolysis of single purified proteins. The possible role of lysosomal enzymes in turnover in vivo was discussed.


Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.


Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.


The photochemical inactivation of peptidyl transferase activity.

The photochemical oxidation of the 50-S ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivation exhibits a maximum at pH 7.5. Erythromycin at a low concentration (0.04 mumol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect. The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.


Properties of the high-molecular-weight protein (spectrin) from human-erythrocyte membranes.

The high-molecular-weight protein component from human erythrocytes has been isolated and its solubility properties studied. In physiological solvent conditions the spectrin is not aggregated and is unaffected, both in hydrodynamic properties and conformation, as judged by circular dichroism and intrinsic fluorescence, by the addition of calcium ions. When the pH is decreased an opalescence first sets in, which corresponds to an associated fibrous state of the protein, and when a critical pH is reached precipitation ensues. The precipitation profile is characterised by extreme sharpness, of the kind observed in the phase separation of polyacid-polybase mixtures or of polyampholytes. The addition of calcium ions displaces this precipitation edge towards higher pH. Sodium ions have a similar but smaller effect. The position of the profile is significantly displaced in aged spectrin preparations, or those from frozen erythrocyte ghosts. Fresh preparations of spectrin consist predominantly of a component sedimenting at 9.7 S, with a minor component at 4.4 S (and traces of higher aggregates). The pattern is independent of the ionic strength, or of the presence or absence of calcium ions. The proportion of the small component increases with time, and in spectrin preparations from frozen ghosts it invariably predominates. At low concentrations of guanidine hydrochloride the larger component gives place progressively to the smaller, and vanishes completely when the concentration of the denaturant reaches 1 M. The two components coexist at concentrations below this, and are not in rapid interconversion equilibrium. On recovery of the protein from the guanidine hydrochloride by dialysis, the original pattern of two components is regained. On the other hand the larger component is not found in the material recovered from guanidine hydrochloride solutions of preparations that contain only the small component at the outset. The recovered spectrin is similar to the starting material in its circular dichroism, in its pH-precipitation profiles, and the manner in which the latter is modified by calcium ions. Molecular weight determination by sedimentation equilibrium shows that the 4.4-S species has a molecular weight of some 230 000, which is also the value derived from the extrapolated sedimentation coeffiecient in 6 M guanidine hydrochloride, and thus corresponds to single chains (of which two or more species are resolved in acrylamide gel electrophoresis in the presence of sodium dodecylsulphate); the 9.7-S species, which characterises what is evidently the native state of the extracted spectrin, is found to be a dimer. The frictional coefficients of the monomer and dimer are appreciably different. That of the dimer is compatible with a somewhat asymmetric structure, but by no means to the extent expected for a myosin-like or paramyosin-like molecule.


Control of haemoglobin synthesis. The effects of iron deprivation, cobalt and temperature on the rate and extent of globin synthesis in reticulocytes.

A detailed examination of the kinetics of protein synthesis in rabbit reticulocytes in the presence of the iron chelating agent 2,2'-dipyridyl showed that between 30 degrees C and 42 degrees C there were characteristically two distinct phases of protein synthesis. An initial phase (I), in which no inhibition of protein synthesis was apparent, was followed by a gradual decline in the rate of protein synthesis leading to the second phase (II) in which protein synthesis occurred at a linear but inhibited rate for extended periods. In contrast, below 30 degrees C, incubation in the presence of dipyridyl caused no inhibition of protein synthesis. Between 30 degrees C and 42 degrees C the duration and amount of protein synthesis occurring in phase I before the onset of inhibition were inversely related of the inhibition as was the final rate of incorporation in phase II. During phase II, a partial reversal of the inhibition caused by dipyridyl was obtained by lowering the incubation temperature. This resulted in a burst of protein synthesis at the uninhibited rate until the amount of protein synthesis reached the same level as that in reticulocytes maintained continuously with dipyridyl at the lower incubation temperature. This burst of synthesis was observed in reticulocytes which had been held in phase II for as long as 90 min. It was also possible to reverse the inhibition by addition of haemin to cells in phase II. At any particular incubation temperature, a fixed number of rounds of protein synthesis had to occur before the onset of phase II became apparent. By the use of puromycin we showed that this was not a requirement for the synthesis of globin or of any other protein. We believe that this critical amount of protein synthesis reflects the residual ability of reticulocytes to initiate new protein chains in the absence of concurrent haem synthesis. Reticulocytes preincubated in the presence of cobaltous ions showed almost no inhibition of protein synthesis upon subsequent incubation with dipyridyl. The results are compared to those obtained in reticulocyte lysates and are discussed in terms of current theories to account for control of protein chain initiation by haemin.


Co-fractionation of an endonuclease activity during the purification of DNA polymerase-alpha from regenerating rat liver. Properties and separation from DNA polymerase.

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high-molecular-weight DNA polymerase-alpha from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co-fractionated with DNA polymerase in a great variety of purification procedures involving ion-exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA-cellulose. The endonuclease acted on native or denatured DNA by introducing single-strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.


Patulin biosynthesis: the metabolism of m-hydroxybenzyl alcohol and m-hydroxybenzaldehyde by particulate preparations from Penicillium patulum.

The ring hydroxylation of m-hydroxybenzyl alcohol to gentisyl alcohol by a particulate preparation from Penicillium patulum has been characterised. The activity was shown to be closely associated with, but not necessarily identical to, m-cresol 2-hydroxylase activity of the 105 000 X g microsomal fraction. As with both the m-cresol hydroxylases of this system, m-hydroxybenzyl alcohol hydroxylase required oxygen and NADPH for activity. A Km value for m-hydroxybenzyl alcohol of 15 muM was measured. Inhibition of the hydroxylase activity and its reversal by light, as well as the action of cytochrome c, KCN and other effectors suggested a mixed-function oxidase reaction of the cytochrome P-450, NADPH-cytochrome reductase type. m-Hydroxybenzaldehyde was not ring hydroxylated by any preparation from P. patulum. Apart from the previously described conversion to m-hydroxybenzyl alcohol by a predominantly soluble dehydrogenase, m-hydroxybenzaldehyde was metabolized to m-hydroxybenzoic acid by a particulate fraction. This activity required NADPH. It was concluded that the main biosynthetic pathway to patulin must be through m-hydroxybenzyl alcohol, gentisyl alcohol and gentisaldehyde.


Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.


Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.


Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes.

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.


The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.


Active enhancement of heart transplants in the inbred rat with donor strain blood.

Prolongation of survival time of heart transplants in inbred rats following intravenous injection of donor strain blood according to various time-dose schedules is described. The immunological status of long-term survivors shows detectable antidonor antibodies, normal graft-versus-host reactions and rejection of skin grafts in the usual time. Lymphocytotoxic activity is well related to the survival time of the heart in those rats which respond to the treatment.


Observations on the effect of CO2 and temperature on the sickling phenomenon.

The explanation of the effect of CO2 on the sickling phenomenon has, in the past, been based solely on the alteration of the oxygen affinity of haemoglobin induced by the CO2, and hence solely on the oxygen saturation of the haemoglobin. A re-investigation of this phenomenon, reported here, shows that this explanation is inadequate; rather, a full explanation requires the use of Perutz's hypothesis that the haemoglobin molecule can exist in various conformationally distinct states, the relative concentrations of which are dependent not only on the oxygen saturation of the molecules but also on other factors such as pH and 2,3-diphosphoglycerate (DPG) concentration. The effect of temperature on the sickling phenomenon is explained in a similar fashion. Low temperature is found to have no effect on the morphology of irreversibly sickled cells.


Changes in potassium content of erythrocytes during exercise in man.

Factors affecting erythrocyte K+ content and plasma electrolytes during light exercise were examined in 8 normal subjects and 27 patients with chronic obstructive lung disease. The patients were classified into bronchitis, emphysema and intermediate groups. Arterial blood was obtained during rest, after 2, 5, and 7 min. of exercise on a bicycle ergometer (30 km/h without mechanical brake), and at 10 and 20 min. during recovery for analysis of PO2, PCO2, plasma H+, Na+, K+, and Cl(-), erythrocyte K+ content and whole blood lactate. Plasma H+ increased significantly in all subjects after 2 min. of exercise and PCO2 rose in normal, bronchitis, and emphysema groups during exercise, whereas PO2 did not change significantly. Plasma K+ rose promptly after the beginning of exercise and remained at high values during exercise. The rapid rise in PCO2 was associated with a significant decrease of calculated erythrocyte K+ in patients with chronic bronchitis. Although changes in plasma H+ were dependent on both PCO2 and lactate, erythrocyte K+ changes were significantly related only to changes in PCO2. These results indicate that the decrease in erythrocyte K+ is mainly controlled by changes in arterial PCO2 during light exercise in patients with chronic bronchitis.


The effect of propranolol and phentolamine on serum gastrin concentration in response to respiratory acidosis in normal man.

Serum gastrin concentration and basal acid secretion were studied in normal subjects under the influence of respiratory acidosis induced by CO2 rebreathing. During the intragastric instillation of 100 ml/h 0.5 M bicarbonate a significant increase of gastrinaemia from 133 to 158 pg/ml (p less than 0.01) occurred in ten subjects during respiratory acidosis (pCO2 62 torr, pH 7.25). Under the intragastric instillation of 100 ml/h 0.1 N HCl the rise of gastrin concentration in response to CO2 rebreathing (pCO2 68 torr, pH 7.20) was not significant. The relationship between the decrease of pH and the increase of the gastrin concentration was shifted in the direction of a greater systemic acidosis compared to the results performed in the presence of a neutral intragastric pH. 50 mug/kg propranolol intravenously produced a decrease of gastrin concentrations from 145 to 127 pg/ml (p less than 0.01) and a total suppression of hypergastrinaemia in response to CO2 rebreathing, suggesting activation of beta-cell receptors in respiratory acidosis. The infusion of phentolamine in a dose of 0.6 to 1.8 mg/min. resulted in a rise of gastrin concentration from 140 to 165 pg/ml (p less than 0.01) which was not further elevated during respiratory acidosis. The basal acid secretion showed a significant rise in response to CO2 rebreathing, which was abolished by the administration of propranolol.


The role of histamine in burn, tourniquet and endotoxin shock in mice.

The release of histamine and mortality was studied in mice after various types of experimental shock. In burn shock, serum histamine rose significantly after injury, but there was no correlation between increased serum histamine and high mortality as a consequence of several therapy regimens. For example, after treatment with histamine or Compound 48/80 before burning, there was a rise of serum histamine, yet shock mortality fell significantly. Although separate administration of antagonists of H1 - or H2 - histamine receptors had no effect on mortality, pretreatment with both diphenhydramine and burimamide significantly increased shock mortality. In tourniquet shock, serum histamine rose significantly, and treatment with both antagonists before trauma produced a significant elevation of shock mortality. In endotoxin shock, prior treatment with one or both drugs did not change mortality. These results suggest that endogenous histamine is not a lethal factor in burn and tourniquet trauma, but rather it appears to have a compensatory, beneficial effect.


Centrally induced hypotensive effects of beta-adrenergic blocking drugs.

The mechanism of the antihypertensive effects of propranolol, pindolol and sotalol were investigated using chloralose-anesthetized cats and dogs. When administered i.v. into a carotid or vertebral artery, propranolol was readily taken up by several brain areas. Highest drug concentrations were detected in the hypothalamus and its projection areas and low concentrations in the cerebral cortex and peripheral tissues. Discrete drug administration at several brain sites decreased efferent sympathetic but increased efferent parasympathetic nerve activity. These neural changes were associated with reductions in both blood pressure and heart rate. Hippocampal propranolol administration induced the greatest neural and cardiovascular changes. Significant concurrent reductions in blood pressure, heart rate and cardiac contractile force occurred following propranolol (500 mug) administration into the hippocampus and pindolol (80 mug) administration into the septum. Sotalo injections at various brain sites failed to influence the cardiovascular system. The centrally induced cardiovascular and neural changes induced by both propranolol and pindolol were prevented by pretreatment with hexamethonium. These results suggest that the hypotensive effects of both propranolol and pindolol may be related to a reduction in the level of central  autonomic control resulting in decreased levels of blood pressure, heart rate and cardiac contractile force.


Neuroleptic antagonism of dyskinetic phenomena.

The bilateral intrastriatal administration of dopamine to guinea pigs pretreated with nialamide-induced dyskinesias characterized by gnawing, biting and licking reactions, acute twisting of the head and neck, whole body or head and neck rocking movements, and a hyperactive state. Hyperactivity was inhibited by the peripheral administration of large doses of many neuroleptic agents, e.g. haloperidol, fluphenazine, but only pimozide and oxiperomide inhibited the other forms of dyskinetic movements. This was not due to a peculiar depressant effect of these agents in the guinea pig since haloperidol was shown to be equipotent to oxiperomide and of greater potency than pimozide both in causing catalepsy and antagonising amphetamine sterotypy...


Pharmacology of nadolol (SQ 11725), a beta-adrenergic antagonist lacking direct myocardial depression.

SQ 11725 was approximately 1/3 as potent as propranolol in blocking the stimulant effects of isoproterenol in vitro, but was 2-4 times more potent than propranolol in blocking the diastolic blood-pressure and heart-rate responses, respectively, to isoproterenol in vivo. SQ 11725 had no depressant effects on contractile force of isolated guinea pig atrial muscle at the largest bath concentration studied (1000 mug/ml), whereas propranolol caused significant depression at a concentration of 1-10 mug/ml. Propranolol was at least 20-30 times more depressant than SQ 11725 to canine myocardium in vivo. SQ 11725 was more effective then propranolol in inhibiting tachycardic responses to treadmill exercise in unanesthetized dogs. The duration of blockade of the heart-rate response to exercise or to isoproterenol administration in the unanesthetized dog was approximately 5 times greater with SQ 11725 than with propranolol.


ACTH-like peptides and opiate receptors in the rat brain: structure-activity studies.

The present study aims at further identifying the interaction of ACTH-like peptides and rat brain opiate receptors in vitro. The sequence ACTH4-10 is crucial with respect to affinity since it is the shortest sequence to inhibit the binding of [3H]-dihydromorphine and [3H]-naltrexone to these receptors. A second active site seems to be localized in the N-terminal part of ACTH11-24. This structure-activity relationship is compared to that observed for these peptides on the adrenal cortex and behavior.


[Characteristics of the combined action of fentanyl and sodium oxybutyrate on respiration].

Experiments conducted with rabbits (86) and albino rats (28) demonstrated that with the use of sodium oxybutyrate (200 mg/kg) in combination with phentanyl (0,02 mg/kg) the former reduces the depressing influence of the latter on the external respiration and tissue processes.


[Effect of sodium oxybutyrate, phenamin, transamine and L-dopa on the processes of work capacity recovery under conditions of minimal rest].

The action of sodium oxybutyrate, phenamine transamine and L-DOPA on the processes of re-establishing the mental and physical performance capacity after fatigue was studied in experiments with rats. The activating effect of sodium oxybutyrate on the resotration of performance capacity in these conditions is shown not to be inferior to an analogous action of phenamine. Complete re-establishment of performance capacity is recorded against the background of the action produced by a monoaminoxidase inhibitor-transamine, as well as combinations of sodium oxybutyrate and L-DOPA.


Ergoline derivatives. Note XIII (1) alpha-adrenergic blocking drugs.

The synthesis and the pharmacological activities of 24 analogues of the alpha-adrenergic blocking drug, nicergoline (I b), are reported. The majority of the new compounds were found to be less active than (I b): the structure-activity relationships are presented and discussed.


[Antiviral chemotherapeutic agents. XVIII. Adamantane derivatives of amphetamine. Their potential interest as autonomic and antiparkinson agents].

Adamantane isologs of amphetamine, methamphetamine and pheniprazine and several derivatives were synthesized in order to study the influence of their more pronounced lipophilic characteristics on their biological properties. A preliminary examination of their toxicity, antiviral, CNS stimulant and antiparkinson activity is described. The adamantyl amphetamine, which proved active, will be further studied.


[Effect of steroid hormones on the rate of NADPH and NADH oxidation by rat liver microsomes].

Sex hormones heighten the rate of the NADP-H oxidation. A relation between the oxidation rate of steroids and the polarity of their molecule has been found. Both androgens and estogens inhibit the transport of electrons in the NAD-H-dependent chain. The capability of steroids to hydrooxylate depends upon the chemical structure and not the affiliation to androgens or estrogens.


[Pharmacological and radioprotective properties of some gamma-pyrone derivatives (flavanones and flavanols)].

In experiments set up on mice, rats and guinea pigs 8 gamma-pyron derivatives were studied. They proved to be little toxic, do not change significantly the arterial pressure and respiration, exercise a depressing action on the central nervous system, produce a marked antiphlogistic (normal and irradiated animals), radio-protective and antihistaminic effects and influence norephinephrine, homovanillic acid and dopamine metabolism in the brain.


Use of 1,3-butanediol for lactation and growth in cattle.

These studies have been designed to test whether 1,3-butanediol (BD) alleviates milk fat depression in lactating cows, to observe physiological changes in blood and rumen constituents when BD is fed to cows or growing cattle, and to test the effects of BD on growth rates and feed efficiency in growing cattle. In trials with lactating cows, milk fat percentage and total fat production were higher for cows fed BD than for controls. Feeding BD to either cows or growing cattle had no consistent effect on rumen pH or relative concentrations of rumen volatile fatty acids. 1,3-Butanediol feeding had little effect on blood glucose concentrations. Feeding more than 4% BD in diets sometimes caused increased concentrations of blood ketones. In trials where growing cattle were fed 4% BD, rates of gain and feed efficiency were at least as good as and often better than those of cattle fed the same diets without BD. Body composition was not significantly affected. 1,3-Butanediol can be utilized effectively as an energy source for cattle and causes no obvious problems with 4% in diets.


Prolonged survival of skin allografts in rats treated with antigen and hydrocortisone and the trend in graft-versus-host reactions.

The mechanism of prolongation of skin allograft survival in rats treated with specific antigen and hydrocortisone was studied in the strain combination with the strong (H-1 + non-H-1) and weak (non-H-1) antigenic difference. The grafts differing from the recipients at the weak non-H-1 loci only showed the longest survival although unresponsiveness to the strong (H-1) antigens of the donor type had already disappeared. In both strain combinations, immunological reactivity of lymphoid cells from treated animals was demonstrated in local GVH reactions at the end of the treatment (i.e., 12 days after transplantation). Lymphoid cells isolated from long surviving (100 days) AVN recipients of non-H-1 different grafts did not react in the GVH test. Attempts to obtain prolongation of skin allograft survival by passive transfer of serum removed after termination of immunization with strong (H-1 + non-H-1) or only weak (non-H-1) antigens were not successful.


Insulin degradation by isolated fat cells and their subcellular fractions.

Isolated frt cells and purified subcellular fractions of fat cells have been shown to degrade insulin to biologically inactive trichloroacetic-acid-soluble fragments. Further study of this activity has revealed the following characteristics: 1 Most of the insulin-degrading enzymes are intracellular, inaccessible to insulin or trypsin when fat cells are intact. More that 90 per cent of the recovered activity is found in the high-speed supernatant (cytosol) when cell fractionation studies are performed. 2. The plasma membrane contains significant insulin-degradative capacity, as shown by tryptic digestion of intact cells and cell fractionation. 3. The pH optimum of the cell-membrane insulin-degrading site is more acid than that of the cytosol activity, but the tow enzyme systems are similar with regard to substrate specificity, response to metabolic inhibitors, and elution volume of degradation products on gel filtration. 4. The plasma-membrane-degrading activity differs from the specific insulin-binding site with regard to saturation kinetics, optimum temperature, substrate specificity, sensitivity to sulfhydryl-blocking agents, and trypsin snesitivity.


Behavioural effects of anti-epileptic drugs.

There is increasing evidence that various types of behavioural disturbance, other than those described for phenytoin, for example, may occur from the use of anti-epileptic medication, and it has been suggested that these disturbances may not always be reversible. There are suspicions also that certain groups of patients--especially those with structural brain damage--are particularly susceptible. These and other factors relevant to the study of the effects of anti-epileptic drugs on behaviour are comprehensively reviewed. It is concluded that the psycho-pharmacology of anti-epileptic drugs is unsatisfactory at present because of inadequate and unsophisticated reporting of behavioural change and because of the common problem of polypharmacy, and that more accurate observations are needed on less heterogeneous groups of patients that have been described in the past.


Amino acid decarboxylase enzymes--vital or irrelevant to gastric secretion?

The location and discovery of histidine decarboxylase and aromatic L-amino acid decarboxylase in the stomach are described. Feeding and gastrin-like agents stimulate increased gastric histidine decarboxylase (HD) acttvity in rats. Procedures which result in increased gastrin release - often by raising antral pH - also have this action in intact but not in antrectomised rats. Evidence for a histaminic feedback mechanism controlling HD levels is discussed. HD activity is reduced by protein synthesis inhibitors and by chronic pyridoxine deficiency. The effects of inhibitors of HD on gastric secretion in rat, dog and man are reviewed. The role of HD and histamine in gastric secretion is considered.


Role of gamma-glutamyl transpeptidase activity in the diagnosis of hepatobiliary disease.

The enzyme gamma-glutamyl transpeptidase is widely distributed throughout the body, notably kidney, seminal vesicles, pancreas, liver, spleen and brain. Being one of the enzymes of the gamma-glutamyl cycle, it is involved in aminoacid transport, catalysing a transpeptidation reaction between gamma-glutamyl peptides and most common amino acids. Methods of assay of the enzyme are based on its ability also to act on synthetic amides of glutamic acid; kinetic methods monitoring the release of p-nitroaniline from the substrate L-gamma-glutamyl p-nitroanilide are the most satisfactory. In diseases of the liver, the highest levels occur in association with cirrhosis, alcoholism, hepatic secondaries and cholestasis. As the enzyme is present in the endoplasmic reticulum of the hepatocyte, its activity is increased in situations leading to microsomal enzyme induction. Raised levels can also occur in pancreatitis, diabetes, myocardial infarction, congestive cardiac failure, chronic renal failure, cerebrovascular accidents, cerebral tumours and chronic obstructive pulmonary disease. Although the lack of specificity must be recognised, the estimation can be useful in the elucidation of some clearly defined problems arising during investigation of patients with suspected hepatic disease, especially where performed as part of a biochemical profile.


Localization of 5'-ribonucleotide phosphohydrolase in regenerating (and normal) limb tissues of the adult newt Notophthalmus viridescens.

The regenerating forelimb of the adult newt, Notophthalmus viridescens was investigated for 5'-nucleotidase (5' ribonucleotide phosphohydrolase, 3.1.3.5) acitivity. The newt's humeri were surgically removed, and after a twenty-one-day recovery period, the forelimbs amputated above the elbows. Regenerates were sampled at predetermined times for specific phases in the progress of regeneration, frozen, sectioned in a cryostat, and the sections fixed in 10% cold formol calcium. The Wachstein and Meisel [25] lead procedure at neutral pH was used predominately in these experiments, although tests were also conducted with Gomori's [14] calcium, Allen's [21] highly alkaline procedures. The substrates used to obtain specific enzyme reactions were adenine, cytosine, guanine, uracil and inosine 5'-monophosphate nucleotides. Sodium beta-glycerophosphate served as a non-specific phosphomonoesterase substrate, distilled water replaced substrate, and inhibitors such as zinc and cyanide ions were used as control measures to assist in increasing the precision in interpreting the results obtained. The most reactive 5'-nucleotidase (5'-Nase) loci were in the walls of the blood vascular system, mysial and neural sheaths, dermis, and periosteum: the principal cells involved were macrophages, endothelium of blood vessels, and fibrocytes of connective tissues. A moderate enzyme response was elicited from secretory cells of some of the subcutaneous glands, hypertrophied chondrocytes and osteogenic centers, chondrocytes in the articular regions and within red blood cells and leucocytes. Normal, injured and degenerating, or regenerating striated muscle and nerve fibers were judged unreactive for 5'-Nase. The epidermis and wound epithelium displayed negative responses for 5'-Nase. Cells forming the regeneration blastema were 5'-Nase reactive during the early formative phase, but with growth and development of the blastema into bulb and conic forms, these cells did not respond for this enzyme-activity. One suggestion offered is that the absence of 5'-Nase in cells of the blastema may be related to the lack of an adequate blood-vascular supply. Several functions of 5'-Nase in normal and regenerating tissues are discussed. A basic conclusion reached is that 5'-nucleotidase hydrolyses may be more involved in fundamental anabolic than in catabolic metabolism.


Vascular permeability-increasing action of hypoalbuminemic substance from Ehrlich ascites carcinoma cells.

A hypoalbuminemic substance (F-3), which was obtained from Ehrlich ascites carcinoma cells, showed a marked vascular permeability-increasing action in mice. The highest increasing effect was produced 20 min after an intradermal injection of F-3. This action was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and indomethacin but not by hydrocortisone or D-2-bromolysergic acid diethylamide. The inhibition of F-3 by diisopropyl fluorophosphate was observed in partial even after the excess diisopropyl fluorophosphate was removed from the diisopropyl fluorophosphate-treated F-3. The hypoalbuminemic and hypoproteinemic actions were not subjected to any inhibition by the diisopropyl fluorophosphate treatment. The decrease in albumin produced 3 hr after the injection of F-3 would be due to the leakage of albumin from blood vessels. F-3 might contain at least two components, one being a vascular permeability-increasing substance and the other a hypoalbuminemic one.


Induction of ornithine decarboxylase, tyrosine aminotransferase, and thymidine kinase by glucocorticoid in isolated, perfused liver after tumor inoculation.

A method for producing solid tumors in rat liver or spleen by local inoculation of Yoshida sarcoma or Hirosaki sarcoma was developed by careful selection of rat strains. After development of the tumor, the liver was isolated and perfused with a mixture of calf serum and fluorocarbon. Addition of corticoid hormone to the perfusion fluid induced tyrosine aminotransferase in normal tissue of the liver and to a lesser degree in the tumor tissue. Corticoid did not cause any detectable induction of thymidine kinase in normal tissue of the liver, but caused slight but definite induction of the enzyme in the tumor tissue. Ornithine decarboxylase was induced in the normal tissue by perfusion with serum alone, even without corticoid, but no enzyme induction was observed in the tumor tissue. The low level of this enzyme found in solid tumor tissue might be due to the fact that the enzyme was measured in the late period of tumor growth, because, in experiments with ascites tumor cells, higher enzyme activities were observed in the early period of growth.


Aryl hydrocarbon hydroxylase in liver nuclei of C3H/He and DBA/2 mice.

Aryl hydrocarbon hydroxylase activity in liver nuclei from C3H/He mice is apparently increased by the administration of 3-methylcholanthrene, but the enzyme activity from the DBA/2 mice is not. On the other hand, by treatment of the mice with phenobarbital sodium, the increased activity in liver nuclei was observed in both strains of mice. There are approximately the same levels of the apparent Km for benzo[alpha]pyrene in liver nuclei from both strains of mice even after treatment with 3-methylcholanthrene, but different Km values for NADPH and NADH are observed between the constitutive and induced enzyme, showing 0.032 and 0.091 mM for NADPH, and 0.303 and 1.67 mM for NADH, respectively. Both 5, 6- and 7, 8-benzoflavones enhance the activity in the constitutive enzyme, but inhibit it in the induced enzyme non-competitively. Nicotinamide inhibits both enzyme activities in liver nuclei. Cyclohexene oxide and 1, 1, 1-trichloropropane oxide enhance the activity in the induced enzyme, but not in the constitutive enzyme in liver nuclei. The differences in the properties between the contitutive and induced enzymes and between the enzymes in microsomes and in nuclei from mouse liver were discussed briefly.


Serum cobalt-activated acylase and gamma-glutamyl transpeptidase activities in toxic hepatitis.

Marked activity of cobalt-activated acylase was found in the sera of 33 of 37 patients with acute toxic hepatitis due to poisoning with either amanita mushrooms or chemicals. The activity of the enzyme showed a positive correlation with that of serum transaminases, reached the highest levels on the patient's admission to hospital and within a few days fell rapidly to undetectable levels. Slight acylase activity was observed in the majority of patients intoxicated with drugs or carbon monoxide but was not seen in sera of those poisoned with non-amanita mushrooms who showed no signs of liver injury. Unlike acylase, the serum activity of gamma-glutamyl transpeptidase remained unchanged over the first days of acute toxic hepatitis. The determination of serum cobalt-activated acylase might be of value in the diagnosis of acute liver injury.


[Studies on the stearic acid dehydrogenase in the liver and brain of rats of various ages (author's transl)].

Tissue slices from the liver and brain of 7-day-old rats incubated with [1-14C]stearic acid desaturate the stearate to oleate. The activities of the two tissues are different but of the same order of magnitude. With increasing age, the activity in the liver increases markedly, while the brain activity decreases. The postmitochondrial supernatant from adult (3-month-old) liver contains 2 to 3 orders of magnitude more stearoyl-CoA dehydrogenase activity than the brain postmitochondrial fraction. The washed microsomal fraction from liver had about the same activity as the postmitochondrial supernatant, but no dehydrogenase activity could be detected in the washed microsomal fraction from the brain. The acyl-CoA synthetase and the palmitoyl-CoA hydrolase activities measured in the washed microsomes from adult brain were both lower than in liver microsomes. The concentration of stearoyl-CoA (the substrate for the stearoyl-CoA dehydrogenase) resulting from the ratio of these activities was too high, however, for the lack of desaturase activity to have been simulated by lack of substrate.


Reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum. Effect of ligands to transition metals on the activity and the stability of the enzyme.

Reduced ferredoxin: CO2 oxidoreductase (CO2-reductase) from Clostridium pasteurianum catalyzes the reduction of CO2 to formate at the expense of reduced ferredoxin, an isotopic exchange between CO2 and formate in the absence of ferredoxin, and the oxidation of formate to CO2 with oxidized ferredoxin. The three activities were found to be equally affected by monovalent anions known to be ligands to transition metals: The enzyme was reversibly inhibited by azide (Ki = 0.004mM), cyanate (Ki = 0.3 mM), thiocyanate (Ki = 1mM), nitrite (Ki = 0.4mM), nitrate (Ki = 6mM), chlorate (Ki = 3mM), fluoride (Ki = 5mM), and by chloride, bromide, iodide (Ki greater than 5mM). There was no observable effect of pH on the inhibition constants. The enzyme was not inhibited by carbon monoxide. The enzyme was irreversibly inactivated by low concentrations (10muM) of cyanide. The rate of inactivation increased with increasing pH with an inflection point near pH 9.5. Reduced ferredoxin and formate rather than oxidized ferredoxin or CO2 protected the enzyme from inactivation by cyanide. The enzyme was protected by azide and cyanate from inactivation. In the presence of high concentrations of the monovalent anions the rate of inactivation by heat (55 degrees C), by molecular oxygen, and by cyanide was decreased by a factor of more than 100. Half maximal protection was observed at the Ki concentrations of the two reversible inhibitors. The data are interpreted to indicate that a transition metal of weak "a class" character and a disulfide are catalytically significant groups of CO2-reductase from C. pasteurianum.


Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.


[15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization].

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.


[IMP-cyclohydrolase/transformylase from ehrlich-ascites carcinoma (author's transl)].

The IMP-cyclohydrolase/transformylase enzyme was purified from Ehrlich ascites tumor cells by ammonium sulfate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex. The electrophoretically pure enzyme has a molecular weight of about 350000, estimated by a gel filtration, and an isoelectric point of 6.2. It is composed of 8 subunits with a molecular weight of 46000. Every subunit is composed of two different proteins with a molecular weight of 18000 and 28500. Some further characteristics of the enzyme are reported.


Criteria of viability of isolated liver cells.

36.4 +/Various cellular parameters were measured with regard to their usefulness as criteria of viability of isolated cells. Stainability by trypan blue and release of lactate dehydrogenase indicate only severe irreversible damage of cells. Neither endogenous respiration nor even the ATP/ADP ratio is a sensitive criterion of viability. On aging of cells, the ATP/ADP ratio remains high, even though the membrane potential, the intracellular K concentration and the content of adenine nucleotides decrease considerably. A sensitive, easily performed test is the stimulation of cellular respiration by 1mM succinate. Only a damaged plasma membrane allows succinate permeation of a rate sufficient to stimulate respiration. The membrane potential and the intracellular Na and K concentrations are the most sensitive criteria of viability, since they indicate the earliest changes on aging. (For freshly isolated cells, we found a membrane potential of 36.4 "/- 3.4 mv [n = 5], an intracellular K concentration of 109.0 +/- 9.1 mM, and an intracellular Na concentration of 47.0 +/- 13.4mM.) The incorporation of [14C]uridine also sensitively reflects cellular damage.


Membrane effects on hepatic microsomal glucose-6-phosphatase.

1) Rat liver microsomes exhibit only a weak hydrolyzing activity towards galactose 6-phosphate. Disruption of the microsomal vesicles does not change the apparent Michaelis constant for this substrate but enhances the apparent maximum velocity. 2) The inhibition of microsomal glucose-6-phosphatase (EC 3.1.3.9) by galactose 6-phosphate is of the competitive type in intact and disrupted microsomal vesicles, suggesting that both substrates are hydrolyzed by the same enzyme. 3) The high degree of latency found for the hydrolysis of galactose 6-phosphate compared to glucose 6-phosphate indicates the presence of a carrier for glucose 6-phosphate in the microsomal membrane. 4) Since glucose as a product is not trapped inside the microsomal vesicles, this sugar probably is able to penetrate the microsomal membrane.


A ligand-induced conformational change of yeast dipeptidase at physiological pH: kinetic consequences and possible regulatory significance.

The hydrolysis of dipeptides by purified yeast dipeptidase (EC 3.4.13.?) shows marked deviations from Michaelis-Menten kinetics over a wide range of pH. Quite anomalous kinetics is observed between pH 6 and 7, indicating a drastic change in the enzyme's properties. A reasonable explanation is provided by the assumption of a conformational transition brought about either by pH shifts or, at a constant pH, by changes in the substrate concentration. The transition, which may have a half-life on the order of minutes under appropriate conditions, is a distinctly cooperative process, with a dependence on ligand concentration higher than first order. The two forms of the enzyme differ clearly from each other with respect to various properties. The magnitudes and pH dependence of the kinetic parameters as well as the type of inhibition (or activation) exerted by amino acids and other ligands are different, as are their heat stabilities and the rates of inactivation by photooxidation of proteolytic  degradation. Neither the molecular weight nor the gross conformation of the enzyme changes during the transition, so it seems to be due to a local isomerization affecting mainly the geometry of the active site. The sensitivity of dipeptidase to changes in the concentrations of substrates and other ligands is most pronounced exactly at the values of pH known to prevail in the living yeast cell. Thus the observed effects, which modulate dipeptidase activities within wide and limits, according to the amounts of dipeptides and amino acids present, are likely to play a role in the regulation of the enzyme in vivo.


The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.


Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

The glucosyltransferase (UDP-glucose galactosylhydroxylsine collagen glucosyltransferase, EC 2.4.1.?.) was purified 50-fold from calf arterial tissue by ammonium sulfate precipitation, gel filtration and electrofocusing. The purified enzyme has a molecular weight of 72 000 and a requirement for Mn2. It resolves into two activity peaks when submitted to electrofocusing (isoelectric point at pH 4.2 and 8.1) or disc electrophoresis and exhibits a double pH optimum (pH 8.3 and 9.9). The enzyme was found to transfer glucose from UDP-glucose to the denatured forms of citrate-soluble calf skin collagen (I), the alphal chain (II) and the beta12 component (III) derived from it, and of an acetic-acid-souble collagen preparation (IV) obtained from alkali-treated calf arterial tissue. The Km values for the substrates were 1.67 X 10(-4) (I), 6.3 X 10(-4) (II), 3.3 X 10(-4) (III) and 2.8 X 10(-4) mol/l (IV), indicating that the enzyme has the greatest affinity for the calf skin collagen. The glucose transferred to hydroxylysine-linked galactose residues may be released subsequently by the action of a specific alpha-glucosidase purified from bovine spleen. The results support the assumtion that the glucosylation step in the course of the (pro-)-collagen biosynthesis depends on special structural features of the substrate and may be controlled by a specific alpha-glucosidase.


Evidence for different glycohydrolase and glycosyltransferase activities of beta-N-acetylglucosaminidases A and B.

1) Two forms of beta-N-acetylglucosaminidase--known as form A and form B - were purified from bovine spleen homogenates and efficaciously separated by preparative disc electrophoresis on polyacrylamide gel. Studies on the enzymatic specificity revealed that the two forms have different glycoside hydrolase and glycosyl transferase activities towards substrates of natural origin. 2) With the trisaccharide GlcNAc-GlcUA-GlcNAc from hyaluronate as substrate, form A released free N-acetylglucosamine at a rate 35-40 times higher than form B. The B form, however, transferred N-acetyl-[6-3H]glucosamine from phenyl-beta-N-acetyl-D[6-3H]glucosaminide to the tetrasaccharides GlcUA-GalNAc-4-sulfate-GlcUA--GalNAc-4-sulfate or GlcUA-GlcNAc-GlcUA-GlcNAc isolated from chondroitin 4-sulfate or hyaluronate at rates 5-10 times higher than beta-N-acetyl-glucosaminidase A, the corresponding 3H-pentasaccharides being isolated as reaction products. 3) The pH optimum of the glycoside hydrolase activity is 4.5, while optimum glycosyl transfer proceeds at pH 6.5. Under condition optimum for glycoside transferase, hydrolytic activity is still observed with each form, but the B form exhibits about equal glycoside hydrolase and glycoside transferase activity, whereas the A form has a predominant glycoside hydrolase action.


Gd(--) Abrami: a deficient G-6PD variant with hemizygous expression in blood cells of a woman with primary myelofibrosis.

A new deficient G-6PD variant, Gd(--) Abrami, was found in granulocytes, platelets and red blood cells of a 65-year-old woman with myelofibrosis. Enzyme and immunological titrations showed that only the deficient variant was present in blood cells whereas both the normal and abnormal enzymes were found in the fat cells of this patient. These results seem to indicate that the granulocytes, platelets and erythrocytes of this woman with myelofibrosis have arisen from a single abnormal precursor the functional X chromosome of which is the one carrying the abnormal G-6PD gene.


Comparative studies on the human glutamate-pyruvate transaminase phenotypes--GPT 1, GPT 2-1, GPT 2.

The quantitative differences between the activity of the 3 common phenotypes of human red cell GPT has been confirmed. In addition, the activity of red cell GPT 1 was found to be greater in young children than in adults. No such difference was found for the GPT 2 phenotype. The activity of the red cell GPT 1 was found to decrease with age, reaching the adult level at the age of 10 to 12 years. Red cell GPT of all the 3 common phenotypes in both adults and children was found to show a similar response to the addition of excess pyridoxal phosphate. A method has been devised for the partial purification of human GPT (cytoplasmic) from liver. GPT 1 and GPT 2 have been purified, and very few significant differences were found amongst the physical and kinetic parameters tested.


[Virus infections of the respiratory tract in childhood (author's transl)].

Comprehensive epidemiological studies revealed that every individual is suffering from a respiratory tract infection in the average 6 times a year. 97% of these are caused by viruses. Identical clinical symptoms may be produced by many different agents, thus the etiology of an illness can only be identified by virus isolation and serological tests. The frequency of isolations is varying in adults and in children as well as in ambulatory and in hospitalized patients. The persistently observed susceptibility for new infections is caused by the great variety of possible etiologic agents especially in upper respiratory infections and the peculiarities of local immunity in the respiratory tract. An influence of chilling could not be demonstrated scientifically in spite of controverse clinical observations. It was proved however that allergic individuals succumb more frequently to viral infections. Overweight in infancy increases susceptibility for bronchitis and bronchiolitis. Superinfections by bacteriae are not rarely complications of viral diseases and antibiotic therapy may become necessary eventually. Leucocytosis and an elevated erythrocyte sedimentation rate are only criteria of limited value to distinguish viral and bacterial infections. Specific virostatic therapy is not possible so far for respiratory tract illnesses, but for prophylaxis of infections with influenza A2 amantadine-HCl may be used. Gammaglobulin has a prophylactic effect as well for certain virus infections but is of little use for antiviral therapy.


Chicken (Gallus gallus) hemolytic complement: optimal conditions for its titration.

The effect of pH, ionic strength, cation concentration, ethylenediaminetetraacetic acid trisodium salt (Na3EDTA), time and temperature were studied to determine the optimal conditions for titrating the hemolytic complement (C) activity in sera of chicken (Gallus gallus). Swine erythrocytes (E) sensitized with rabbit antibodies were the most sensitive, while chicken serum had a smaller amount of "natural" antibody against them than against red blood cells from other five species tested. The highest titers of chicken C, when tested with swine sensitized E, were detected when isotonic NaCl-barbital buffer was used as diluent, having the ionic strength of 0.15, conductance of 11 millimhos/cm at 20 degrees C. However, maximal chicken C titers detected with sensitized rabbit E were obtained at ionic strength of 0.07 to 0.11 depending on pH. A final concentration of 1 X 10(-3) M of Mg2+ and 3 X 10(-4) M of Ca2+ and pH 8 were optimal in both cases. The temperature of 30 degrees C and time of 60 minutes were appropriate to reveal the maximal titers..


Purification of soluble complement-fixing antigens from two Burkitt's lymphoma cell lines. Conditions affecting the stability and recovery of antigens and antibodies.

Soluble Epstein--Barr virus (EBV) associated antigens of P3HR-1 and Raji cell lines, which fix complement in the presence of human sera containing antibody against EBV--VCA antigens, were partially purified by fractionating centrifuged cell lysates by the method of immunoadsorbents or by precipitation with ammonium sulphate or glycine--HCl. Purification conditions were limited by the instability of both antigen and antibody activity. The antibody was inactivated under all the conditions commonly used to dissociate antigen--antibody complexes. The antigen was inactivated at pH 2-3, and was irreversibly bound to Sephadex G-200 conjugates which had not been exposed to large excesses of serum.


[Changes in the acid-base equilibrium in hypokalemic patients during potassium infusions with various anions].

Based on the parameters of the acid-base status, the effect of different anions in potassium and magnesium substitution was studied. A mixture of chloride with the metabolisable anions acetate and malate does not cause any changes in the acid-base balance. The exclusive supply of metabolisable anions in the form of aspartate always results in a change of blood pH values towards an alkalosis.


[Acid-base equilibrium- -disorders and therapy].

The purpose of this work is to give a brief resume, with figures and tables, of the knowledge concerning the acid-base balance which is indispensable in intensive therapy. The effects of disturbances of the acid-base balance on organic functions are shown and, therefore, the importance of a situation-adapted therapy is particularly stressed. In this connection, the role of infusion therapy in the treatment of disturbances of the acid-base balance is especially discussed.


[Systematic and random errors in the determination of parameters of the acid-base equilibrium].

A review is given of the most common errors made in the estimation of the parameters of acid-base balance. Especially important are the systematic errors, for instance in handling the blood before estimation, by use of incorrect standard solutions or poor temperature control during the measurement of the pH. By paying attention to these details, it is possible to obtain excellent precision in final measurement in the standard apparatus.


Barriers to the nurse practitioner movement: problems of women in a woman's field.

Barriers to the nurse practitioner movement are explored in this article. Historically, the subordination of women and the sex segregation of nursing and medicine have helped to establish interaction patterns between the two professions which included a subordination of nurses as well as informal doctor-nurse games. These patterns were reinforced by hospital training schools and state laws which restricted the roles of nurses. Recent changes in state movement, but the stereotyped communication patterns between nurses and physicians remain as a barrier to the full use of the talents of nurses in practitioner roles.


The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function.

A new amino protecting group, the methylsulfonylethyloxycarbonyl (Msc) group, is described which combines well with other familiar groups (benzyloxycarbonyl, t-butyloxycarbonyl) in peptide syntheses. Its main characteristics are an extreme acid stability, a high base lability and a high polarity which enhances solubility in polar solvents including water. The group resists catalytic hydrogenolysis but does not poison the catalyst. It has good crystallizing properties. Application in peptide synthesis is exemplified in the synthesis of Msc-Phe-Arg-Trp-Gly-OMe.HCl. Deprotection of the masked tetrapeptide was accomplished within 5 sec with a 1.0 N solution of base (OH- and OCH3-). A reaction scheme for the cleavage of the group is suggested.


Radiation inactivation of rabbit muscle aldolase.

Pulse radiolysis and steady-state X-radiolysis have been used to investigate the radiation inactivation of aldolase from rabbit muscle. Both eaq-and OH readily react with aldolase, and contribute to inactivation. The radical anions (CNS)2-and (Br)2-react with aldolase at neutral pH. The progressive addition of alkali results in an increase in the second-order rate constants, with an apparent pK approximately 10 +/- 0-3, and with the formation of an unstable intermediate, lambdamax approximately 400 nm resembling a phenoxyl radical. Steady-state radiolysis in the presence of (CNS)2-and (Br)2- at alkaline pH results in increased aldolase inactivation, with a pK of enzyme inactivation similar to that observed for reaction of the radical anions. We propose that a reaction of the radical anoins with tyrosine residues accounts for the resultant inactivation.


Folic acid conjugase from plasma. III. Use of the enzyme in the estimation of folate activity in foods.

A convenient method for the estimation of food folates using human plasma as a source of folic acid conjugase has been standardized. Bovine, goat and dog plasma could not be used for the purpose because of low enzyme activity and slow reaction rates. Human plasma conjugase released food folates from conjugated forms over a pH range of 4.5 to 6.0. The food folate values obtained by employing rat liver and chicken pancreas conjugase closely agreed with the values obtained by using human plasma conjugase. The advantages in using human plasma as a source of conjugase were (1) easy availability (2) non-essentiality of preliminary processing (3) stability of the enzyme on storing plasma in cold (4) low enzyme blank folate activity and (5) low amount of plasma needed for assay. Using this method, folic acid activity of some common Indian foods of plant and animal origin has been estimated. There were wide variations in the folate activity of different classes of foods. In general legumes and green leafy vegetables were found to be rich in folate activity.


Standardized aerobic and anaerobic exercise: differential effects on intraocular tension, blood pH, and lactate.

The effects of standardized aerobic and anaerobic exercise intensities on intraocular tension, blood lactate, and pH were studied. Intraocular tension decreased rapidly at all exercise intensities. The absolute lowest level of intraocular tension reached with aerobic and anaerobic exercise levels varied by only 1.5 mm. Hg and this difference was not statistically significant. Blood lactate and pH changes correlated with intraocular tension changes at anaerobic exercise levels, but not at aerobic exercise levels. These findings associated with aerobic exercise have not been previously reported. It is suggested that parameters other than the decrease in blood pH and the increase in blood lactate are responsible for most of the decrease in intraocular tension associated with dynamic exercise.