bluuebunny/crossref_metadata_2025 huggingface dataset:
Deoxyribonucleic acid reiatedness among species of Haloarcula and other halobacteria
Authors: Gutierrez M. C. , Ventosa A. , Ruiz-Berraquero F.
Published: December 2009

Several representative strains of Haloarcula vallismortis, Haloarcula hispanica, and other Haloarcula species not currently accepted as valid species were examined for genetic relatedness to each other and to members of other halobacterial species. The G + C content of DNA of the 11 strains of the genus Haloarcula tested ranged between 56.9 and 66.3 mol%. The G + C contents obtained for Haloarcula vallismortis and Haloarcula hispanica were 56.9–64.3 mol%, and 58.7–66.3 mol%, respectively. The results of our DNA–DNA hybridization experiments indicate that Haloarcula vallismortis and Haloarcula hispanica are composed of strains with high DNA homology, constituting genotypic species that correlate with the previously reported phenotypic features. However, our results do not support the separation of "Haloarcula californiae," "Haloarcula sinaiiensis," "Halobacterium marismortui," and "Amoebobacter morrhuae" in different taxonomic taxa, since high DNA homology was obtained between these strains and representatives of species of the genus Haloarcula.Key words: archaebacteria, halobacteria, Haloarcula, DNA homology, nucleic acids hybridization.
Biosynthesis of phosphatidylglycerol and phosphatidylglycerolphosphate in submitoehondrial membranes isolated from guinea pig liver is absolutely dependent on CDP-diglycerides imported from microsomal membranes
Authors: Stuhne-Sekalec Lidija , Stanacev Nikola Z.
Published: December 2009

The biosynthesis of radioactively labelled phosphatidylglycerol via phosphatidylglycerophosphate in outer and inner mitochondrial membranes isolated from guinea pig liver was found to depend absolutely on CDP-diglycerides, which could not be biosynthesized in these membranes. The requirement for CDP-diglycerides in the biosynthesis of labelled phosphatidylglycerol could be fulfilled by the transfer of biosynthesized [3H]CDP-diglycerides from the microsomal membranes to the outer and inner mitochondrial membranes.Key words: submitoehondrial membranes, transfer, CDP-diglycerides, phosphatidylglycerol, phosphatidylglycerophosphate.
Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida
Authors: Buckley J. Thomas
Published: December 2009

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.Key words: aerolysin, protease, activation, purification, Aeromonas.
Inhibition of rat perirenal preadipocyte differentiation
Authors: Roncari Daniel A. K. , Blanc Paul E. Le
Published: December 2009

The process of adipose differentiation uniquely endows fat cells to accrue triacylglycerols under conditions of nutrient energy surfeit and to release fatty acids during energy deprivation. The object of this investigation was to study influences on this process in perirenal preadipocytes, grown in primary culture or first subculture and derived from male Sprague–Dawley rats, 180–200 g. Supplementation of the culture medium with 1-methyl-3-isobutylxanthine, corticosterone, and insulin induced differentiation in practically all perirenal preadipocytes, as indicated morphologically and by rising glycerophosphate dehydrogenase activity. Appreciable differentiation was induced even in the absence of methylisobutylxanthine. Transforming growth factor β (1–1000 pM), cachectin (tumour necrosis factor α) (1–1000 pM), and basic fibroblast growth factor (0.063–63 nM) inhibited adipose differentiation significantly, almost completely at the higher concentrations. Direct inhibition, rather than a persisting mitogenic effect of fibroblast growth factor, was confirmed using demecolcine (Colcemid). The fact that transforming growth factor β and cachectin inhibit differentiation in preadipocytes from postpuberal rats suggests that this effect probably also occurs in vivo, thus diverting energy from adipose depots in certain neoplastic and inflammatory states. We propose that the anterior pituitary, through fibroblast growth factor(s), modulates the pool of preadipocytes and other mesenchymal cells. The mitogenic effect would be complemented by a concerted function, inhibition of adipose differentiation, resulting in the retention of a greater number of potentially replicative cells. Then, depending on the subject's nutritional and endocrine status, extrapituitary factors would regulate the specific process of differentiation.Key words: preadipocyte differentiation, inhibition, pituitary, cachectin.
The influence of K+-induced membrane depolarization on insulin secretion in islets of lean and obese (ob/ob) mice
Authors: Fournier Linda A. , Heick H. M. C. , Bégin-Heick Nicole
Published: December 2009

The present study was undertaken to determine whether factors that affect K+ permeability produce differences in insulin secretion in the islets of obese versus lean mice. At basal glucose (3 mM), the obese islets secreted more insulin for a given increment in depolarizing K+ concentration and responded to a wider range of K+ concentrations (5–45 mM) than the lean islets (5–25 mM). In contrast, the membrane potential changes induced by increments in pK+ were not significantly different in the two types of islets. The islets of lean and obese mice treated with pertussis toxin showed a qualitatively similar response to glucose and to epinephrine, but only the control and pertussis toxin treated obese islets responded to K+ depolarization when deprived of calcium. Abnormal responses to quinine and apamin were identified in the islets of obese mice. These findings show that the abnormal insulin secretory response of the obese islet is due, at least in part, to a defect independent of glucose metabolism. This is best explained by an altered sensitivity of voltage-dependent events, most likely the result of differential effects of an intracellular element acting on ATP-sensitive and Ca2+-activated K+ channels, both of which are implicated in membrane repolarization.Key words: pertussis toxin, voltage-activated Ca2+ channels, ATP-sensitive K+ channels, membrane potential.
Oxidation of 2-oxoisocaproate and 2-oxoisovalerate by the perfused rat heart. Interactions with fatty acid oxidation
Authors: Letto Joan , Brosnan John T. , Brosnan Margaret E.
Published: December 2009

The interactions between fatty acid oxidation and the oxidation of the 2-oxo acids of the branched chain amino acids were studied in the isolated Langendorff-perfused heart. 2-Oxoisocaproate inhibited the oxidation of oleate, but 2-oxoisovalerate and 2-oxo-3-methylvalerate did not. This difference was not attributable to the magnitude of the flux through the branched chain 2-oxo acid dehydrogenase, which was slightly higher with 2-oxoisovalerate than with 2-oxoisocaproate. Oxidation of 2-oxoisocaproate in the perfused heart was virtually complete, since more than 80% of the isovaleryl-CoA formed from 2-oxo[1-14C]isocaproate was further metabolized to CO2, as determined by comparing 14CO2 production from 2-oxo[14C(U)]isocaproate with that from the 1-14C-labelled compound. Only twice as much 14CO2 was produced from 2-oxo[14C(U)]isovalerate as from the 1-14C-labelled compound, indicating incomplete oxidation. This was confirmed by the accumulation in the perfusion medium of substantial quantities of labelled 3-hydroxyisobutyrate (an intermediate in the pathway of valine catabolism), when hearts were perfused with 2-oxo[14C(U)]isovalerate. The failure of 2-oxoisovalerate to inhibit fatty acid oxidation, then, can be attributed to the fact that its partial metabolism in the heart produces little ATP. We have previously shown that 3-hydroxyisobutyrate is a good gluconeogenic substrate in liver and kidney, and postulate that 3-hydroxyisobutyrate serves as an interorgan metabolite such that valine can serve as a glucogenic amino acid, even when its catabolism proceeds beyond the irreversible 2-oxo acid dehydrogenase in muscle.Key words: branched chain amino acids, branched chain 2-oxoacids, perfused heart, fatty acid metabolism, 3 -hydroxyisobutyrate.
Mitochondrial translocation and processing of the precursor to the α-subunit of rat liver succinyl-CoA synthetase
Authors: Majumdar Ramanath , Bridger William A.
Published: December 2009

Succinyl-CoA synthetase functions in the mitochondrial matrix as an αβ-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (λSCS19) that apparently encodes the cytoplasmic precursor to the α-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature α-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific α-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.Key words: succinyl-CoA synthetase, mitochondria, protein translocation, signal sequence.
Stereospecific analysis of menhaden oil triacylglycerols and resolution of complex polyunsaturated diacylglycerols by gas–liquid chromatography on polar capillary columns
Authors: Myher J. J. , Kuksis A. , Yang L.-Y.
Published: December 2009

The sn-1,2-, sn-2,3-, and X-1,3-diacylglycerols derived by Grignard degradation of purified menhaden oil triacylglycerols were isolated by conventional thin-layer chromatography with boric acid complexing. The sn-1,2(2,3)-diacylglycerols were resolved into sn-1,2- and sn-2,3-diacylglycerols by stepwise digestion with phospholipase C of the corresponding phosphatidylcholines and the positional distribution of the fatty acids were determined. Diacylglycerols were converted into trimethylsilyl ethers and resolved on the basis of molecular weight and degree of unsaturation by gas–liquid chromatography using a polar capillary column and isothermal or programmed temperatures. The order of chromatographic elution was established for more than 70 major and minor species by reference to primary and secondary diacylglycerol standards and by calculation of relative retention times. The identified molecular species ranged in carbon number from 28 to 44 and in double bond number from 0 to 12 being made up of C14–C22 fatty acids with 0 to 6 double bonds each and representing the n – 9, n – 6, n – 4, n – 3, and n – 1 series. The gas–liquid chromatographic determinations yielded proportions of all major species that were consistent with those calculated from the knowledge of the stereospecific distribution of the fatty acids in the original triacylglycerol molecules.Key words: Grignard degradation, rac-phosphatidylcholines, phospholipase C, enantiomeric diacylglycerols, thin-layer chromatography, molecular species of diacylglycerols, composition of fatty acids.
The phase behaviour of lipid X at high water concentration
Authors: Lipka G. , Hauser H.
Published: December 2009

The phase behaviour of lipid X at high water concentrations (> 60%) is studied using mainly differential scanning calorimetry (DSC) and X-ray diffraction. The critical micellar concentration (CMC) decreases from 8 × 10−5 M at −2 °C to 4 × 10−5 M at 20 °C. The critical micellar temperature (CMT) is 0 °C and decreases slightly with increasing lipid X concentration. Above the CMC and below the CMT, lipid X forms a lamellar gel phase (Lβ). Above 0 °C and at concentrations ranging from the CMC up to about 0.2 M (20%), lipid X forms small micelles. At even higher concentrations there is a transition to a hexagonal phase, probably hexagonal I. Addition of excess NaCl to lipid X dispersions at concentrations < 0.2 M (20%) has several effects on the phase behaviour of lipid X. (i) The lamellar phase is stabilized up to temperatures of ≈20 °C at [NaCl] ≥ 0.7 M. (ii) NaCl induces a tighter packing of the hydrocarbon chains. (iii) At concentrations > 0.7 M NaCl, the bilayer repeat distance decreases to about 43 Å (1 Å = 0.1 nm).Key words: lipid X, phase behaviour, effects of NaCl, differential scanning calorimetry, X-ray diffraction.
Analyse lipidique en taxonomie bactérienne: proposition d'une méthode standardisée
Authors: Asselineau Cécile , Asselineau Jean
Published: December 2009

To increase the application of lipid analysis for taxonomic purposes, a standardized method would be useful. This method has to be simple enough to be used routinely. Such a method, based on thin-layer and gas chromatography, is proposed. It can be applied to any kind of bacteria when a few milligrams of cells are available. Comparison of the results provided by this analysis with data found in the literature (given in a schematic form in six tables) allows the identification of the studied strain in the most favorable cases, or gives information for the choice of conclusive complementary tests in the other cases.Key words: taxonomy, lipids, bacteria.
Hormonal responses of glucose-6-phosphatase catalytic unit studied by stopped-flow analysis
Authors: Traxinger Roger R. , Nordlie Robert C.
Published: December 2009

To obtain insight regarding the mechanism(s) of response of the catalytic unit of glucose-6-phosphatase (D-glucose-6-P phosphohydrolase; EC 3.1.3.9) to glucocorticoid administration and insulin deprivation, the functional enzyme concentration E0 was estimated from presteady-state kinetics by the stopped-flow technique. The E0 values were compared with Vmax values determined by the steady-state kinetic approach. Studies were carried out with detergent-disrupted microsomes from livers of normal fed, 48-h fasted, streptozotocin-diabetic, and triamcinolone-treated rats. All of the treatments caused an increase in E0, but Vmax values were increased only in fasting and diabetes. Km values were unaffected by all the treatments. The increase in Vmax observed with fasted and diabetic rats was explained by an increase in E0 alone. These results showed that insulin deprivation resulted in an increased formation of fully active glucose-6-phosphatase catalytic unit. In contrast, administration of triamcinolone caused an increase in E0 but not in Vmax. It was concluded that glucocorticoid administration may promote formation of catalytic units of glucose-6-phosphatase which are less active than the enzyme normally present or formed in response to insulin deprivation.Key words: glucose-6-phosphatase, glucocorticoids, diabetes, stopped-flow analysis.
Characterization of an endogenous quail intestinal lectin
Authors: Fang Rixun , Ceri Howard
Published: December 2009

Soluble extracts of quail intestine scrapings contain a lectin activity specific for chicken and rabbit trypsinized, glutaraldehyde-fixed erythrocytes. The lectin displayed a specificity for the simple sugar haptens lactose and galactose and for mucin. Quail lectin was purified by affinity chromatography on either asialofetuin- or mucin-Sepharose, followed by DEAE-Sepharose chromatography, and demonstrated an apparent molecular weight of 14 500 on sodium dodecyl sulfate – polyacrylamide gel electrophoresis and a pi of 6.2 upon isoelectric focusing. Immunohistochemical localization of this lectin in the intestine was carried out using polyclonal antibody raised in rabbits and tested for specificity in Western blots. Immunoperoxidase staining for quail lectin showed the lectin to be prominent in secretions at the mucosal surface and in goblet cells.Key words: endogenous lectin, intestinal, mucin, goblet cells.
High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli
Authors: Shi Wen , Chow King-Chuen , Wong J. Tze-Fei
Published: December 2009

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-β-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be detectible on the basis of complementation testing.Key words: tryptophan-tRNA, aminoacyl-tRNA synthetase, Bacillus subtilis.
Mobilization of arachidonic acid in thrombin-stimulated human platelets
Authors: Mahadevappa V. G. , Sicilia Frank
Published: December 2009

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.Key words: deacylation, phospholipids, thrombin, platelets, phospholipase A2.
Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs
Authors: Yao Li-Juan , Richardson Carol , Ford Carol , Mathialagan N. , Mackie George , Hammond Geoffrey L. , Harding Paul G. R. , Possmayer Fred
Published: December 2009

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5′ and 3′ regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5′ end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5′ region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3′ end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3′ fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5′ and 3′ ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea – sodium dodecyl sulphate –polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-β-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small. These studies demonstrate the feasibility of producing mature human SP-B through recombinant DNA technology, but indicate that it may be necessary to use a fusion protein approach to obtain sufficient amounts for the formation of artificial pulmonary surfactant.Key words: pulmonary surfactant (human), hydrophobic proteins, artificial pulmonary surfactant, in vitro translation.
Stimulation of poly(ADP-ribose) synthesis by free radicals in C3H10T1/2 cells: relationship with NAD metabolism and DNA breakage
Authors: Lautier Dominique , Poirier Danièle , Boudreau Annie , Alaoui Jamali Moulay A. , Castonguay André , Poirier Guy
Published: December 2009

We have studied the effect of H2O2 and O2− produced by xanthine and xanthine oxidase on NAD catabolism, poly(ADP-ribose) synthesis, and production of DNA single-strand breaks in C3H10T1/2 cells. The results show a correlation between the induction of DNA single-strand breaks, the decrease of NAD pool, and the accumulation of polymer. New techniques, based on affinity chromatography and reversed-phase high pressure liquid chromatography, have allowed an accurate determination of polymer contents and showed a 20-fold stimulation of polymer biosynthesis induced by active oxygen species. Inhibition experiments performed with 3-aminobenzamide have shown that the decrease in NAD levels after exposure of cells to active oxygen species was caused by stimulation of poly(ADP-ribosyl)ation and of another cellular process.Key words: poly(ADP-ribose), free radicals, C3H10T1/2 cells, DNA breakage, NAD catabolism.
Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor
Authors: Takagi Shinko , Hummel Brian C. W. , Walfish Paul G.
Published: December 2009

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass ≈ 50 000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 μCi/μg; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 × 109 to 1.74 × 109 M−1 for PTU and 1.79 × 109 M−1 for MMI), while they both decreased (p < 0.02) maximal binding capacity (from 0.36 ± 0.02 to 0.19 ± 0.02 pmol/mg protein for MMI and 0.17 ± 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with ≈25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats. Pretreatment of CNR for 1 h with 5 mM methyl methanethiosulfonate (an oxidant of thiol groups) abolished the inhibitory effects of PTU, MMI, or arsenite, but was not inhibitory in itself. From these studies it is concluded that (i) MMI and PTU could exert at least part of their therapeutic effects by inhibiting specific binding of L-T3 to its hepatic nuclear receptor; (ii) these inhibitory effects of MMI and PTU are likely due to an interaction with cysteine residues (some of which are not in a dithiol configuration) that are essential for T3 binding to its receptor; and (iii) binding of T3 is not inhibited by oxidation of receptor thiols to methyl dithiol groups.Key words: nuclear triiodothyronine (T3) receptor, methimazole, propylthiouracil, Scatchard analysis.
The pH dependence of the reconstitution reaction of apotyrosinase: the question of Cu(I) versus Cu(II)
Authors: Wigfield Donald C. , Goltz Douglas M.
Published: December 2009

The rates of reconstitution of apotyrosinase by CuSO4 at various pH values have been determined. The rate at pH 6.0 is 130 times faster than that at pH 9.0. These results show that the idea of reconstitution by Cu(I) being a faster process than by Cu(II) by a factor of 200 should be discarded.Key words: tyrosinase, copper.
Nucleophilicity and ionization of dihydrolipoamide
Authors: Hunter Christie L. , Danson Michael J. , Stevenson Kenneth J.
Published: December 2009

The reactivity of dihydrolipoamide has been studied at pHs 6 and 8 with a series of haloalkyl acids, iodoacetamide, and N-phenyl-α-bromoacetamide. The electron-withdrawing capability of the R group in these reagents (X—CH2—R) has the dominant influence on their reactivity with the thiolate. Thus, 3-bromopyruvic acid greatly exceeds N-phenyl-α-bromoacetamide, iodoacetamide, iodoacetic acid, and three bromoalkyl acids. A plot of the second-order rate constant of iodoacetamide with dihydrolipoamide supports a value of pK1, in the vicinity of 10.5. The second pKa of dihydrolipoamide cannot be deduced by this method, since reaction of the first thiol with iodoacetamide greatly alters the influences which the vicinal (normally unmodified) thiol has on the other. Potentiometric titration of dihydrolipoamide was not definitive, but the curve obtained was comparable to the titration curve of dihydrolipoamide (followed spectrophotometrically) which accommodated a pK1of 9.35 and pK2 of 10.65. As a control, a plot of the second-order rate constant versus pH for the alkylation of thioglycolic acid revealed a pKa of 10.5 in agreement with the titrated pKa of 10.4.Key words: dihydrolipoamide, nucleophilic addition reactions, ionization of thiols, iodoacetamide.
Dicoumarol-induced 9-γ-carboxyglutamic acid prothrombin: isolation and comparison with the 6-, 7-, 8-, and l0-γ-carboxyglutamic acid isomers
Authors: Malhotra Om P.
Published: December 2009

The role of γ-carboxyglutamic acid (Gla) in prothrombin function can be effectively evaluated by characterizing dicoumarol-induced, Gla-deficient prothrombin structural isomers. In addition to the isolation of 8-, 7-, 6-, 5-, 3-, 2-, 1-, and 0-Gla isomers, we have now purified a variant prothrombin containing 9(8.80) Gla residues by barium citrate adsorption, elution, and finally by DEAE-cellulose and immunoaffinity chromatographies. Agar gel electrophoretic mobilities of the 9-Gla isomer and its fragment 1 were slower than those of the respective 10-Gla (normal) prothrombin and fragment 1, both in the absence and presence of Ca(II). In the presence of Ca(II), both 9- and 10-Gla fragments 1 moved slower than 8- and 7-Gla fragments 1. However, in the absence of metal ions, 9- and 7-Gla fragments 1 migrated at the same rate, but slower than 10- and 8-Gla fragments. Similarly, the 9-Gla fragment 1 electro focused cathodically to 10- and 8-Gla, but comparably with 7-Gla fragment 1. The 9-Gla fragment 1 exhibited a Ca(II)-induced 44% decrease in the intrinsic fluorescence, compared with a 40% decrease in that of 10-Gla; 8-Gla fragment 1 revealed only 23% quenching. Ca(II)-dependent anti-normal prothrombin antibodies are not specific for 10-Gla prothrombin, since only a twofold molar excess of the 9-Gla isomer was required to displace equal amounts of labeled normal prothrombin. The most critical Gla residue for influencing the functional, thrombin-generating properties of prothrombin appears to be the one present in the 9-Gla isomer but absent in the 8-Gla variant, since 9-Gla prothrombin possesses four times the normal coagulant activity (78 versus 20%) of the 8-Gla isomer.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.
The effect of inhibitors of DNA synthesis on 40-kilodalton polypeptide translation in rat L6 myoblasts
Authors: Meadus William J. , Bag Jnanankur
Published: December 2009

Messenger RNA coding for a polypeptide of 40 kilodaltons (P40) was translated in proliferating rat L6 myoblasts but not in the terminally differentiated myotubes. The relationship between DNA synthesis, differentiation, and P40 mRNA translation was studied. Aphidicolin, a reversible inhibitor of DNA synthesis, was shown to block DNA synthesis in proliferating myoblasts without allowing these cells to differentiate. A second inhibitor, cytosine arabinoside, when added to dividing myoblasts also prevented differentiation. In the absence of biochemical differentiation P40 mRNA remained in the translated state. Translational repression of this mRNA was, therefore, linked to the biochemical differentiation of rat L6 myoblasts.Key words: translational control, DNA synthesis, aphidicolin, P40 mRNA, myogenesis.
Studies on the metabolism of glycolyl-CoA
Authors: Vamecq Joseph , Poupaert Jacques H.
Published: December 2009

Glycolyl-CoA can be formed during the course of the β-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this β-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its β-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of γ-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial β-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial β-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial β-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA. ω-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.Key words: glycolyl-CoA, ω-hydroxymonocarboxylates, β-oxidation, 4-hydroxybutyrate, CoA-dependent 3-hydroxypyruvate oxidation, pyruvate dehydrogenase, citrate synthase, hydroxy citrate.
Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of insulin
Authors: Cánepa Eduardo T. , Llambías Elena B. C. , Grinstein Moisés
Published: December 2009

In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of δ-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of δ-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of δ-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.Key words: δ-aminolevulinic acid synthase, ferrochelatase, cAMP, insulin, diabetic rat hepatocytes.
The oligomeric state of spectrin in the rat erythrocyte membrane skeleton
Authors: Avery Ross A. , Bettger William J.
Published: December 2009

The oligomeric state of spectrin in the erythrocyte membrane skeleton of the rat was investigated following extraction in a low ionic strength buffer for 24 and 96 h. All analyses were quantitively compared with preparations from human erythrocyte membranes. After nondenaturing agarose–polyacrylamide gel electrophoresis, the human samples revealed their characteristic spectrin oligomer pattern; there were high molecular weight complexes near the origin of the gel, followed by several high order oligomers, tetramers, and dimers. The pattern in the rat membrane skeleton also included tetramers and a high molecular weight complex band, but had only one oligomer and no dimers. With time the high molecular weight complex diminished and oligomers accumulated in both the rat and human, while dimers accumulated only in the human and tetramers accumulated only in the rat. Tetramers decreased with time in the human. Extraction of spectrin increased with time and was greater from rat than the human red cell membrane at both time points. The percentage of spectrin and actin in the low ionic strength extract was similar between species, as analyzed by SDS–polyacrylamide electrophoresis, staining, and densitometry. Proteins 4.1 and 4.9 were present in greater percentages in the human. The only temporal effect on monomeric protein composition was an increase of protein A in the rat. There was no species difference in protein A percentage at 24 h, but at 96 h the rat was greater than the human. The results suggest that there are significant differences in the structural arrangement of the rat and human erythrocyte membrane skeleton.Key words: spectrin, erythrocyte, membrane, cytoskeleton.
Two stress proteins of the endoplasmic reticulum bind denatured collagen
Authors: Nandan Devki , Ball Eric H. , Sanwal Bishnu D.
Published: December 2009

A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42 °C, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin–Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification.Key words: stress proteins, collagen-binding proteins, endoplasmic reticulum, 78-kilodalton glucose-regulated protein, 46-kilodalton glycoprotein.
Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer
Authors: Robinson John J.
Published: December 2009

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2+-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2+-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2+-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.Key words: hyaline layer, composition, organization, development.
Free fatty acids enhance the oxidation of oxymyoglobin and inhibit the peroxidase activity of metmyoglobin
Authors: Stewart John M.
Published: December 2009

The effect of low concentrations of sodium oleate on the oxidation of oxymyoglobin to metmyoglobin has been examined. This long chain fatty acid results in a tripling of the initial rate (1.5–4.3 h−1) at which oxymyoglobin is converted to metmyoglobin and more than doubling of the rate of the long-term reaction (0.12–0.33 h−1). Examination of rate constant enhancement over a range of oleate concentrations (0–0.215 mM) has allowed an estimate of association constants for both phases of the reaction system. The peroxidase activity expressed by metmyoglobin towards hydrogen peroxide is inhibited by the presence of sodium oleate by a fivefold increase in the apparent Km value (0.33–1.77 mM). The observed changes in oxymyoglobin concentration over time are discussed in terms of competition between metmyoglobin, which acts as a peroxidase decreasing in situ concentrations of H2O2, and oxymyoglobin, which also is oxidized by the peroxide. It is shown that oleate can bind to metmyoglobin and azidometmyoglobin, but not oxymyoglobin. Catalase reduces the oxidation rates of oxymyoglobin in the presence or in the absence of oleate, substantiating the involvement of H2O2. The results are discussed in relation to the potential increase in tissue peroxidations in the presence of ischaemically elevated fatty acid concentrations.Key words: myoglobin, fatty acid, peroxidation, autoxidation, peroxidase.
Control of proteoliposomal cytochrome c oxidase: the overall reaction
Authors: Nicholls Peter , Cooper Chris E. , Wrigglesworth John M.
Published: December 2009

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (ΔΨ) and pH gradient (ΔpH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse ΔΨ and increase ΔpH; the respiration rate decreases. High levels of valinomycin, however, decrease ΔpH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases ΔΨ and collapses ΔpH; the respiration rate increases. On a millivolt equivalent basis ΔpH is a more effective inhibitor of activity than is ΔΨ. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both ΔpH and ΔΨ components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing ΔpH but in a nonlinear way to ΔΨ ("non-ohmically"). High levels of both ΔΨ and ΔpH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both ΔpH and ΔΨ components of the proton-motive force, but is more sensitive to ΔpH than to ΔΨ at an equivalent ΔμH+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella™.Key words: cytochrome c, cytochrome oxidase, proteoliposomes, respiratory control, modelling, valinomycin, nigericin.
Control of proteoliposomal cytochrome c oxidase: the partial reactions
Authors: Nicholls Peter
Published: December 2009

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith–Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by ΔpH and by ΔΨ can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.Key words: proteoliposomes, cytochrome c, cytochrome oxidase, membrane potential, pH gradient, cytochrome a, electron transfer.
Characterization and distribution of bombesin-like peptides in the rat brain and gastrointestinal tract
Authors: Hernanz Angel
Published: December 2009

Extracts of rat brain and gastrointestinal tract, analyzed by reverse-phase high-performance liquid chromatography and radioimmunoassay, contained two bombesin-like immunoreactivity peaks with similar retention times as porcine gastrin-releasing peptide (GRP) and its COOH-terminal decapeptide, neuromedin C or GRP(18–27). However, the GRP-like peptide peak did not elute with exactly the same retention time as porcine GRP. The highest concentration of bombesin-like immunoreactivity was found in extracts of antrum, whereas the lowest was found in whole brain. Neuromedin C was present at lower concentrations than the GRP in antrum, duodenum, and ileum, while similar amounts of each were found in brain.Key words: gastrin-releasing peptide, neuromedin C, rat, brain, intestine.
Melengestrol acetate and megestrol acetate are prostatic tumor inhibiting agents
Authors: Padilla G. M. , Yacullo R. C. , Padilla J. J. , Payne B. , Petrow V.
Published: December 2009

We had previously reported that 6-methylene progesterone, an inhibitor of 5α-reductase, the enzyme which converts testosterone to dihydrotestosterone, markedly inhibited growth of the androgen-dependent Dunning R3327-H rat prostatic tumors. We now find that the progesterone derivatives melengestrol acetate (MGA) and megestrol acetate (MA) inhibit both the androgen-dependent (Dunning R3327-H) and the androgen-independent (Dunning R3327-AT3) prostatic tumors. Growth of the AT3 tumors was suppressed by ~53% after 9 days of daily s.c. injections with MGA at 10 mg/kg body weight. MGA also caused a 54% weight reduction of the ventral prostate and a 53% reduction of the seminal vesicles. Adrenal weights were reduced by 42%. A 24-day oral treatment with MGA (at ~15-17 mg/(kg∙day)) inhibited AT3 tumor growth by 59% and caused a weight reduction in the following tissues: prostate (46%), seminal vesicles (19%), testes (12%), and adrenals (52%). Under the same protocol, MA inhibited AT3 tumor growth by 32% and reduced the weight of the ventral prostate by 49% and the weight of the adrenals by 18%, but had no effect on the seminal vesicles and testes. The extent of the MGA-induced prostatic regression was accompanied by cytological changes similar to those effected by 6-methylene progesterone, i.e., shrinking of the acinar epithelium. The AT3 tumors in MGA-treated rats displayed a limited degree of apoptosis. Atrophy of the adrenal cortex and lowered plasma levels of corticosterone and dihydroepiandrosterone were also observed. A therapeutic role for MGA and MA against androgen-independent prostatic neoplasms in man is forecast by these observations.Key words: prostatic cancer, androgen independent, melengestrol acetate, growth inhibition, steroids.
Oxyradical production results from the Fe3+–doxorubicin complex undergoing self-reduction by its α-ketol group
Authors: Hasinoff Brian B.
Published: December 2009

A variety of different measures have been used to compare the self-reduction of the Fe3+ complexes of doxorubicin and daunorubicin. The Fe3+ –doxorubicin complex exhibited a much faster rate of (i) O2 consumption, (ii) self-reduction under Ar to the Fe2+ complex, (iii) aerobic reduction of ferricytochrome c, (iv) scavenging of Fe2+ by bipyridine, (v) hydroxyl radical production measured by electron paramagnetic resonance spin-trapping experiments, and (vi) inactivation of the cytochrome c oxidase activity of beef heart submitochondrial particles, than did the corresponding Fe3+ –daunorubicin complex. In contrast to Fe3+ –doxorubicin, the Fe3+ –daunorubicin complex displayed only a fast phase of inhibition of the cytochrome c oxidase activity, indicating that the initial binding of these two Fe3+ –drug complexes is very similar. All of these results indicate that Fe3+ –doxorubicin undergoes a much faster self-reduction to the Fe2+ complex and hence a much greater rate of production of damaging oxyradicals when the Fe2+ is reoxidized by O2 or H2O2. The addition of the α-ketol acetol to Fe3+–daunorubicin resulted in greately increased rates of (i) ferricytochrome c reduction, (ii) Fe2+ production, and (iii) hydroxyl radical production. These results support the hypothesis that the α-ketol functional group of doxorubicin (which is not present on daunorubicin and is the only structural difference between these two compounds) reduces the Fe3+ while undergoing oxidation itself.Key words: doxorubicin, adriamycin, iron, self-reduction, oxyradical.