Purification and properties of an S-type pyocin, pyocin AP41

Pyocin AP41, a protease-sensitive bacteriocin produced by Pseudomonas aeruginosa PAF41, was purified to a homogeneous state and characterized. The molecular weight of this pyocin was about 95,000 as determined by the combination of gel filtration and sedimentation velocity analysis. This pyocin was a complex of two kinds of polypeptides. Highly purified preparations showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their apparent molecular weights were 90,000 and 6,000 to 7,000, respectively. Two proteins could be separated by gel filtration in the presence of 6 M urea. Amino acid compositions of these components were determined. The large component had pyocin activity similar to the complex, whereas the small component did not. Sensitive cells were killed by this pyocin only under growing conditions and with single-hit kinetics. The pyocin-treated cells lysed in about 30 min with concomitant production of their resident pyocins or phages. The induced production of resident pyocins caused by pyocin AP41 depended on a recA gene function.     

Monkey pepsinogens and pepsins. III. Carbohydrate moiety of Japanese monkey pepsinogens and the amino acid sequence around the site of its attachment to protein.

Purified Japanese monkey pepsinogens I and II contain carbohydrate as a part of the enzyme molecule. By gel filtration on Sephadex G-100, chromatography on DE-32 cellulose, and polyacrylamide disc gel electrophoresis, the carbohydrate moiety could not be separated from the enzyme protein, and the content did not decrease on repeated chromatography. Glycopeptides were obtained by successive digestion of pepsinogens with thermolysin and aminopeptidases and isolated by chromatography on Sephadex G-25 and G-50. Identification and determination of carbohydrate components was performed by paper and gas-liquid chromatographies. The presence of 4 glucosamines, 6 galactoses, 6--8 mannoses, and 8--11 fucoses per molecule of the glycopeptide of both pepsinogens was observed, of which the high content of fucose is especially unique. The molecular weight of the carbohydrate chains should be around 4,000--5,000. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

Development-dependent expression of cathepsins d and e in various rat tissues, with special reference to the high expression of cathepsin e in fetal liver

The levels of cathepsins D and E in various rat tissues during development were determined with the sensitive assay method we have developed. The level of cathepsin D increased gradually in each tissue during fetal development suggesting the gradual maturation of the lysosomal system in a cell. The level of cathepsin E differed significantly between tissues at various developmental stages. The level in liver increased rapidly from 13-day-gestation fetal stage and decreased gradually at later fetal stages. The level in other tissues such as stomach and spleen began to increase at later fetal stages or the infant stage. Cathepsin E was found in fetal hepatocytes and its gene was hypomethylated when the expression of the gene was elevated. The enzyme was found to be present mainly as a proform suggesting that, after working, an active form is rapidly inactivated.     

Slow Evolutionary Rate of GB Virus C/Hepatitis G Virus

With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 × 10⁻⁶ per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000–10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection. Received: 2 June 1998 / Accepted: 8 August 1998     

Effect of hypertonic saline on the corticotropin-releasing hormone and arginine vasopressin content of the rat pituitary neurointermediate lobe

The changes in the levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the neurointermediate lobe of the pituitary (NIL) following hypertonic saline administration were examined in rats. The plasma osmotic pressure in rats receiving 2% NaCl for 8 days was greatly increased. Plasma AVP concentration in rats receiving 2% NaCl for 8 days were significantly higher than in control rats (566% of the control level). Plasma corticosterone was significantly higher in the saline-treated rats than in controls, whereas plasma ACTH was not significantly different. The pituitary ACTH concentration was much higher in the saline-treated rats than in controls. CRH in the NIL was increased significantly by saline treatment (419% of the control concentration), whereas the CRH in the paraventricular nucleus and median eminence of control and saline-treated rats did not differ significantly. The AVP in the NIL fell greatly in saline treated rats. The extract from both control and saline-treated rats showed a major peak for immunoreactive CRH, with a retention time identical to that of rat CRH. However, the peak was much higher in the extract from saline-treated rats. The immunoreactive AVP peak was greatly reduced in saline-treated rats. These results suggest that hypertonic saline administration increases the CRH in the NIL and causes AVP hypersecretion and/or hyperfunction of magnocellular-NIL CRH might be responsible for pituitary-adrenal stimulation in saline-treated rats.     

Synthetic analogs of growth hormone-releasing factor with antagonistic activity in vitro

Analogs of human and rat growth hormone-releasing factor (hGRF and rGRF), related to [D-Arg2]hGRF(1-29)NH2, were synthesized by solid phase methodology. Their capacity to inhibit growth hormone secretion stimulated by hGRF(1-44)NH2 was tested on rat anterior pituitary cells in monolayer culture. Among the analogs of hGRF, [D-Arg2,29,Arg30]hGRF(1-30)NH2 showed the highest antagonistic potency of 3.64 relative to [D-Arg2]hGRF(1-29)NH2 = 1. However, the most potent analog synthesized thus far was [N-Ac-His1,D-Arg2,Ala15]rGRF(1-29)NH2, which showed a relative potency of 27.7.     

Glutathione S-transferase π localizes in mitochondria and protects against oxidative stress

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST π, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST π is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST π and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST π completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST π. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST π diminished these changes. GST π-targeting siRNA abolished the protective effect of GST π on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST π under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST π protects mitochondria against oxidative stress.     

Development of microsatellite markers for Pythium helicoides

Pseudomonas sp. strain WBC-3 utilizes methyl parathion ( O,O -dimethyl O - p -nitrophenol phosphorothioate) or para -nitrophenol as the sole source of carbon, nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had been characterized previously and found to be located on a typical class I composite transposon that comprised IS 6100 (Tn mph ). In this study, the transposability of this transposon was confirmed by transposition assays in two distinct mating-out systems. Tn mph was demonstrated to transpose efficiently in a random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia sp. U2 by sequence analysis of the Tn mph insertion sites, both exhibiting MPH activity. The linkage of the mph -like gene with IS 6100 , together with the transposability of Tn mph , as well as its capability to transpose in other phylogenetically divergent bacterial species, suggest that Tn mph may contribute to the wide distribution of mph -like genes and the adaptation of bacteria to organophosphorus compounds.