The polypeptide 310-helix as a template and a spacer

Summary X-ray diffraction analyses have provided detailed structural information on the 3₁₀-helices of (i) pBrBz-d-(Me)Phe-(Aib)₂-d-(Me)Phe-Aib-OtBu and Ac-(Aib)₂-l-Lys(Bz)-(Aib)₂-l-Lys(Bz)-(Aib)₂-NHMe as suitable templates for molecular recognition studies, and (ii) pBrBz-TOAC-(l-Ala)₂-TOAC-l-Ala-NHtBu as an appropriate spacer for an ESR study of side chain to side chain interactions. In addition, in Ac-TOAC-(Aib)₂-l-Trp-Aib-OMe, forming a 3₁₀-helix, the TOAC residue plays the role of an effective quencher of the fluorescence of the tryptophan residue located one turn apart.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Nitric oxide synthesis is blocked in the enteral nervous system during dormant periods of the snail<Emphasis Type="Italic"> Helix lucorum</Emphasis>

During dormancy of terrestrial snails, the whole neuromodulation of the nervous system is deeply modified. In this work we studied the adaptation of a previously described, putatively nitric oxide (NO) forming enteral network to the long-term resting periods of the snail Helix lucorum. The standard NADPH diaphorase (NADPHd) technique, which is an accepted method for histochemical NO synthase (NOS) detection, labeled the same enteric neurons of the midintestine in active or hibernated snails. Quantification of the NO-derived nitrite by the Griess reaction established that the nitrite formation is confined to the NADPHd-reactive network containing the midintestinal segment. In active snails, the nitrite formation could be enhanced by the NOS substrate l-arginine (10 M–1 mM), but decreased by the known NOS inhibitors 1 mM N-nitro-l-arginine (NOARG) and 10 mM aminoguanidine (AG). Application of 1 mM l-arginine and 1 mM NOARG decreased the amplitude of the midintestinal muscle contractile activity, but did not affect the rectal motility. In dormancy, the nitrite formation was reduced in the NADPHd-reactive midintestinal network. Application of l-arginine could not provoke nitrite production and did not influence the midintestinal motility. Our findings indicate that NO is involved in the neural transmission to intestinal muscles of gastropods, but enteric release of NO is blocked during dormancy. The decreased NO synthesis is possibly due to an as yet undefined mechanism, by which the l-arginine/NO conversion ability of NOS could temporarily be inhibited in the long-term resting period of H. lucorum.T. Röszer and Zs. Czimmerer contributed equally to this work as lead authors. This research was supported by OTKA grant no. T42762 (G.B.) and PRCH Student Science Foundation grants 2000, 2002 (T.R.) and 2003 (Zs. Cz). The study is dedicated to Borbála Vecsei-Czimmerer, Elemér Czimmerer, Ágnes M. Fodor-Röszer and József S. Röszer.     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

sll1722, an unassigned open reading frame of<Emphasis Type="Italic"> Synechocystis</Emphasis> PCC 6803, codes for <Emphasis Type="SmallCaps">l</Emphasis>-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate synthase

l-myo-Inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to l-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms. Two forms of the enzyme have been characterized from higher plants, viz. cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g. Spirulina sp.), the presumed chloroplast progenitors. The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp. PCC 6803. Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx. 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools. The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS. The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl -d-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification. Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others.Abbreviations CBB Coomassie Brilliant Blue - EST Expressed sequence tag - G6P d-Glucose 6-phosphate - IPTG Isopropyl -d-thiogalactopyranoside - MIPS l–myo-Inositol 1-phosphate synthase - ORF Open reading frame     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^– pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono--lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

Cyclic hexapeptide antagonists of the bradykinin B2 receptor: Receptor binding and solution backbone conformation

Summary Cyclic hexapeptide analogs of bradykinin, based on a folded receptor-bound model of bradykinin, were found to be able to antagonize the action of bradykinin at its B₂ bradykinin receptor. The best of these, cyclo(d-Lys(Arg)-Phe-Ser-d-Tic-Oic- Arg) [compound 17], has affinities at the human and rat B₂ bradykinin receptors of 230 and 8.5 nM, respectively. This potency is significant, since the analogs lack the C-terminal carboxylate group, residues 2–4 and the important interaction of Phe⁵. These constrained analogs may serve as tools for the determination of the receptor-bound conformation of antagonists at the bradykinin receptor and for the design of even smaller and more potent antagonist analogs.Abbreviations Arg(Me) N-methyl-l-arginine - Arg(Me)₂ N,N-dimethyl-l-arginine - Boc t-butoxycarbonyl - Oic (S,S,S)-octahydroindole-2-carboxylic acid - PAM phenylacetamidomethyl - PyBOP benzotriazole-l-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate - Thi -(2-thienyl)-l-alanine - Tic l-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid     

Acidiphilium aminolytica sp. nov.: An acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment

Acidiphilium aminolytica is proposed for a species of the genusAcidiphilium. Acidiphilium aminolytica can be phenotypically differentiated from all other species of the genusAcidiphilium. The seven strains of this species that have been studied are Gram-negative, aerobic, mesophilic, non-sporeforming, motile, and rod-shaped bacteria. They grow between pH 3.0 and 6.0, but not at pH 6.5. They yield positive results in tests for hippuric acid hydrolysis, catalase and urease production. Oxidase, esculin hydrolysis, and -galactosidase tests are negative. They can used-glucose,d-galactose, inositol, sorbitol,l-lysine,l-glutamate,l-arginine, -alanine,dl-4-aminobutyrate,dl-5-aminovalerate, sperimine, or diaminobutane as a sole carbon source, but cannot use elemental sulfur and ferrous iron as an energy source. The DNA base composition is 58.7–59.2 G+C mol%. The major isoprenoid quinone is ubiquinone with ten isoprene unit (Q-10). The major fatty acid is the C_18:1 fatty acid. Two ornithine amide lipids, the C_18:1 fatty acid esters of -N-3-hydroxystearylornithyltaurine and -N-3-hydroxystearylornithine, are detected as the polar aminolipid. DNA relatedness between this species and the other species ofAcidiphilium, the generaAcidomonas, andAcidobacterium was 29 to 2%. These results indicate, that this new species should be placed in the genusAcidiphilium. The type strain (strain 101) ofA. aminolytica is JCM 8796.     

Utilization ofd-amino acids byFusobacterium nucleatum andFusobacterium varium

Summary The utilization ofd- andl -amino acids with acidic, basic or polar side chains was demonstrated by HPLC. Two species of the anaerobeFusobacterium utilized D-lysine and the L isomers of glutamate, glutamine, histidine, lysine and serine. OnlyF. varium usedl-arginine,d-glutamate andd-serine as substrates, whereasF. nucleatum specifically utilizedd-histidine andd-glutamine.d-Glutamate accumulated in F. nucleatum cultures supplemented withd-glutamine, and ornithine was detected when eitherdl- orl-arginine was included inF. varium cultures. Based on literature precedents,d-glutamate andd-histidine are isomerized to their L isomers prior to degradation, but separate catabolic pathways are possible for each enantiomer of lysine and serine.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Structure-activity relationship of a novel peptide substrate for p60c-src protein tyrosine kinase

Summary We recently reported the identification of a peptide (YIYGSFK) as an efficient substrate for p60^c-src using a random combinatorial peptide library screening method. Over 70 analogues of YIYGSFK were designed and synthesized on beads and their phosphorylation on solid phase by p60^c-src was quantitated by the PhosphorImager. A hydrophobic l-amino acid in position 2 and a basic amino acid in position 7 proved crucial for activity as a substrate. In addition, the l-tyrosine residue at position 3 was critical as the phosphorylation site and was found to be stereospecific, as substitution with the d-enantiomer at this position rendered the peptide totally inactive.Abbreviations -alanine - -aminocaproic acid - Ac N-acetyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - Cha l-cyclohexylalanine - Chg l-cyclohexylglycine - Dab l-diaminobutyric acid - Dap l-diaminoproprionic acid - DIEA N,N-diisopropylethylamine - DMF dimethylformamide - Fmoc fluorenylmethyloxycarbonyl - HOBt N-hydroxybenzotriazole - MeF N-methyl-l-phenylalanine - MeG N-methylglycine - MeI N-methyl-l-isoleucine - MES 2-[N-morpholinolethanesulfonic acid - Nle l-norleucine - Orn l-ornithine - TFA trifluoroacetic acid - Z-Sar benzyloxycarbonyl-sarcosine - Z-Tyr benzyloxycarbonyl-l-tyrosine     

Single Taste Stimuli Elicit Either Increases or Decreases in Intracellular Calcium in Isolated Catfish Taste Cells

Taste cells are specialized epithelial cells that respond to stimulation with release of neurotransmitters onto afferent nerves that innervate taste buds. In analogy to neurotransmitter release in other cells, it is expected that neurotransmitter release in taste cells is dependent on an increase in intracellular Ca²⁺ ([Ca²⁺] _i ). We have studied changes in [Ca²⁺] _i elicited by the taste stimuli l- and d-arginine in isolated taste cells from the channel catfish (Ictalurus punctatus). In a sample of 119 cells, we found 15 cells responding to l-arginine, and 12 cells responding to d-arginine with an increase in [Ca²⁺] _i . The response to l-arginine was inhibited by equimolar d-arginine in cells where d-arginine alone did not cause a change in [Ca²⁺] _i , which is consistent with mediation of this response by a previously characterized l-arginine-gated nonspecific cation channel antagonized by d-arginine [31]. However, we also found that these taste stimuli elicited decreases in [Ca²⁺] _i in substantial number of cells (6 for l-Arg, and 2 for d-Arg, n= 119). These observations suggest that stimulation of taste cells with sapid stimuli may result in simultaneous excitation and inhibition of different taste cells within the taste bud, which could be involved in local processing of the taste signal. Received: 25 May 1995/Revised: 29 September 1995     

Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585

Summary We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various -lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicilin N synthase gene of the cephalosphorin pathway.Offprint requests to: J. Piret