αγ-Enolase in the Rat: Ontogeny and Tissue Distribution

Abstract: The rat brain enolases are dimers composed of α and γ subunits. At pH 8.6 αγ-enolase seemed to be stable, and no evidence was found for the possible formation of αγ-enolase from αα-enolase and γγ-enolase in the course of rat brain homogenization. During ontogeny of the rat forebrain, αγ-enolase was formed before γγ-enolase. The half-maximal specific concentrations were reached at postnatal days 14 and 23, respectively. The distribution of αγ- and γγ-enolase in various rat brain areas was also investigated. In all areas both forms were present. In neuroendocrine tissues αγ-enolase was present at a much higher concentration than γγ-enolase. The ratio between γγ-enolase and αγ-enolase may be indicative of the degree of neuronal maturation, a conclusion further substantiated by the high ratio observed in cerebellum and the low ratio observed in olfactory bulbs, both compared with the ratio in forebrain.     

操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

Effect of steroidal glycoalkaloids of the potato on the permeability of liposome membranes

At pH 7.2, the potato glycoalkaloid α-chaconine caused release of entrapped peroxidase from phosphatidylcholine liposomes containing different free sterols but was ineffective against sterol-free liposomes. The alkaloid was able to complex with all the tested sterols in vitro although there was no close correlation between the extent of sterol binding and liposome disruption. α-Solanine also complexed with sterols in vitro but had no effects on sterol-containing liposomes under these conditions. Both sterol concentration and alkaloid concentration were limiting factors in the action of chaconine but did not markedly affect that of solanine. Solanine destabilized liposome membranes only at pH values of 8 and above but was less effective than chaconine. The importance of the carbohydrate moiety of glycoalkaloids was further demonstrated by the inability of β₂-chaconine to complex with sterols or disrupt liposomes.     

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

Synthesis of γ-Glutamyldopamine and Other Peptidoamines in the Nervous System of Aplysia californica

The synthesis of a series of gamma-glutamyl amines (gamma-Glu-amines), including gamma-Glu-dopamine, gamma-Glu-5-hydroxytryptamine, gamma-Glu-octopamine, gamma-Glu-tryptamine, gamma-Glu-tyramine, and gamma-Glu-phenylethylamine, by nervous tissue of the marine mollusc Aplysia californica is described. After ganglia were incubated in vitro with 14C-amines, the unchanged amine and a new 14C-labeled product, identified as the gamma-Glu conjugate of the amine, were isolated from the tissue extracts. Identification was made by comparing the chromatographic properties (HPLC, TLC, and LC) of the isolated conjugates with chemically synthesized gamma-Glu-amines before and after acid hydrolysis.     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Whole genome scan to detect quantitative trait loci for bovine milk protein composition

The objective of this study was to perform a whole genome scan to detect quantitative trait loci (QTL) for milk protein composition in 849 Holstein–Friesian cows originating from seven sires. One morning milk sample was analysed for the major milk proteins using capillary zone electrophoresis. A genetic map was constructed with 1341 single nucleotide polymorphisms, covering 2829 centimorgans (cM) and 95% of the cattle genome. The chromosomal regions most significantly related to milk protein composition ( P _genome < 0.05) were found on Bos taurus autosomes (BTA) 6, 11 and 14. The QTL on BTA6 was found at about 80 cM, and affected α_S1-casein, α_S2-casein, β-casein and κ-casein. The QTL on BTA11 was found at 124 cM, and affected β-lactoglobulin, and the QTL on BTA14 was found at 0 cM, and affected protein percentage. The proportion of phenotypic variance explained by the QTL was 3.6% for β-casein and 7.9% for κ-casein on BTA6, 28.3% for β-lactoglobulin on BTA11, and 8.6% for protein percentage on BTA14. The QTL affecting α_S2-casein on BTA6 and 17 showed a significant interaction. We investigated the extent to which the detected QTL affecting milk protein composition could be explained by known polymorphisms in β-casein , κ -casein , β-lactoglobulin and DGAT1 genes. Correction for these polymorphisms decreased the proportion of phenotypic variance explained by the QTL previously found on BTA6, 11 and 14. Thus, several significant QTL affecting milk protein composition were found, of which some QTL could partially be explained by polymorphisms in milk protein genes.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

The mechanism of liver microsomal lipid peroxidation

In the presence of Fe³⁺ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe³⁺ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe²⁺ by oxygen.     

Characterization of Opioid Receptor Subtypes in Solution

Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.     

Embryo sac development and endogenous gibberellins in pollinated and unpollinated ovaries of walnut (Juglans regia)

Glycosidases were extracted from grapefruit ( Citrus paradisi Macf. cv. Ruby Red) flavedo, albedo, and juice vesicles harvested at five periods throughout the season. Flavedo β-galactosidase activity was high at the September harvest and then significantly declined by November. Thereafter, no further changes occurred in β-galactosidase activity. Flavedo α-galactosidase activity was low and unchanged throughout the study. α-Mannosidase, initially low in flavedo, steadily increased with advanced maturity. Trends in glycosidase activities of albedo were similar but attenuated. Juice vesicle β-galactosidase did not change through the study period, whereas α-galactosidase activity decreased 70% after the initial harvest period. α-Mannosidase was initially high and then decreased to 50% of the original activity. A second peak of activity was measured in March, followed by a second decline. Extractability differences of the glycosidases suggest differences in compartmentation and function. Two isozymes of α-mannosidase were separated in flavedo and one in juice vesicles, and characteristics were determined at an early and late harvest period. The results suggest that changes in the three glycosidases could be used to further define maturity and senescence in grapefruit.     

Guanine Nucleotide Regulation of [125I]β-Endorphin Binding to Rat Brain Membranes: Monovalent Cation Requirement

The binding of [125I]beta h-endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl-5'-imidodiphosphate. In contrast to the binding of the mu-selective opioid agonist, [3H][D-Ala2,MePhe4,Glyol5]enkephalin, and the delta-selective opioid agonist, [3H][D-penicillamine2, D-penicillamine5]enkephalin, [125I]beta h-endorphin binding was not affected by GTP or guanylyl-5'-imidodiphosphate in a concentration-dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl-5'-imidodiphosphate resulted in a concentration-dependent inhibition of [125I]beta h-endorphin binding. This inhibition was significantly greater than the decrease in [125I]beta h-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]beta h-endorphin binding in the presence of sodium, inhibition of [125I]beta h-endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]beta h-endorphin, the binding of the putative sigma receptor agonist, (+)-[3H]SKF 10,047, was not significantly altered by GTP or guanylyl-5'-imidodiphosphate in the absence or presence of sodium.     

Effects of Various Forms of Stimulation on the Content of Enolase Isozymes and S-100 Protein in Superior Cervical Sympathetic Ganglia Excised from Rats

Contents of the three forms (alpha alpha, alpha gamma, and gamma gamma) of enolase isozymes and S-100 protein in superior cervical sympathetic ganglia (SCG) excised from rats were determined by the sensitive method of enzyme immunoassay, after application of various forms of stimulation, during incubation for 3 h at 37 degrees C in vitro. The amounts of the three forms of enolase isozymes and of S-100 protein in the SCG were not altered by preganglionic or postganglionic stimulation (10 Hz) or by the addition of acetylcholine (1 mM) or a high concentration of K+ (70 mM) to the incubation medium. Norepinephrine (NE; 50 microM), as well as isoproterenol (200 microM) or 3,4-dihydroxy phenylethylamine (dopamine; 200 microM), increased the ganglionic alpha alpha and alpha gamma enolase content to 1.5 to 2.0 times the control level, whereas NE tended to slightly decrease the gamma gamma enolase content. The increase in the alpha isozymes did not appear until after 2 to 3 h of incubation with this agent as a result of an increase in protein synthesis. Propranolol, an adrenergic antagonist, partly inhibited the NE-induced increase in both alpha alpha and alpha gamma enolases. NE and its agonists also considerably increased the S-100 protein level in the SCG; however, the effect developed within half an hour of incubation as a result of the conversion of the bound S-100 protein to the water-soluble form, and did not greatly increase thereafter. Cyclic AMP (1 mM) produced the same kind of increase in the ganglionic S-100 protein content as NE did.(ABSTRACT TRUNCATED AT 250 WORDS)     

The same gamma-secretase accounts for the multiple intramembrane cleavages of APP

It has been hypothesized that different C-terminus of beta-amyloid peptide (Abeta) may be generated by different gamma-secretase activities. Recently, we have identified a new zeta-cleavage site at Abeta46, leading to an important finding that the C-terminus of Abeta is produced by a series of sequential cleavages. This finding prompted us to examine the effects of the known gamma-secretase inhibitors on different steps of the gamma-secretase-mediated sequential cleavages and specifically their effects on the formation and turnover of the intermediate Abeta(46). Our results demonstrate that some of the known inhibitors, such as L-685,458 and III-31C as well as inhibitors IV and V, inhibit the formation of secreted Abeta(40/42) by inhibiting the formation of the intermediate Abeta(46). However, most of the other inhibitors show no inhibitory effect on the formation of the intermediate Abeta(46), but rather inhibit the turnover of Abeta(46), resulting in its accumulation. In addition, the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and sulindac sulfide have no effect on the formation and turnover of Abeta(46), but rather modulate the ratio of secreted Abeta at a step after the formation of Abeta(40) and Abeta(42). Thus, our data strongly suggest that the multi-sequential intramembrane cleavages of amyloid precursor protein C (APP) are likely catalyzed by the same gamma-secretase.     

Coinduction of α-L-arabinofuranosidase and α-D-galactosidase formation in Trichoderma reesei RUT C-30

Abstract Formation of α-L-arabinosidase can be induced in Trichoderma reesei by growing the fungus on L-arabinose or dulcitol, and by adding L-arabinose, L-arabitol, D-galactose, or dulcitol ot non-growing mycelia. The same conditions also stimulated the formation of α-D-galactosidase, but not that of various other enzymes involved in hemicellulose degradation. The optimal inducer concentration with all compounds was 4 mM for both enzymes. Using L-arabinose and D-galactose, the induction efficiency was highest at pH 6.5, whereas induction by arabitol and dulcitol was more efficient at low pH (2.5). The addition of 50 mM glucose did not repress α-L-arabinosidase or α-D-galactosidase formation. These findings suggest coregulation of two hemicellulose side-chain cleaving enzymes in T. reesei .     

Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.