Relative role of low- and high pressure reflexes on sympathetic activity in humans during simulated gravitational stress

In order to determine the relative role of low- and high-pressure reflexes, respectively, on forearm sympathetic nerve activity (fSNA), 10 normal male subjects underwent a 4-step (5 min each) graded lower body negative pressure (LBNP) from -10 to -50 mmHg. Central venous pressure (CVP) and stroke volume gradually decreased (p<0.05), and arterial pulse pressure (PP) abruptly decreased at LBNP of -50 mmHg. Mean arterial pressure (MAP) remained unchanged. Forearm venous plasma norepinephrine concentration (fvNE) increased significantly at LBNP of -35 mmHg (p<0.05) and with a further sharp increase during LBNP of -50 mmHg (p<0.05). High degrees of intra-individual correlations were observed between changes in Log [fvNE] and CVP (r-values from -0.78 to -0.96, p<0.01). We conclude that low-pressure reflexes are the major determinants of fSNA during non-hypotensive gravitational stress (MAP and PP unchanged). When the gravitational stress is more pronounced, a decrease in PP further augments fSNA through inhibition of high-pressure arterial baroreflexes.     

Phylogenetic relationships among transposon-like elements in human and primate DNA

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence. Correspondence to: G.B. Petersen     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

BBReader: A computer program for the combined use of the BioMagResBank and PDB databases

A computer program (BBReader) was developed which performs an inverse search in theBioMagResBank database. Given (cross) peak positions of a protein, the program searchesfor atoms with matching chemical shifts and suggests possible assignments for user-specifiedhomo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments.It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilizedfor filtering possible NOESY cross peak assignments.     

Novel cyanoguanidines with potent oral antitumour activity

4-Pyridyl cyanoguanidines with hydrophobic aromatic side chains showed potent antiproliferative activity in the human breast and lung cancer cell lines MCF-7, NYH and H460. In vivo, treatment with N-(6-chlorophenoxyhexyl)-N′-cyano-N″-4-pyridylguanidine (18, 20 mg/kg/day po.), gave a complete remission of tumours in a model of NYH inoculated nude mice.     

Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation

Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.