Peptidase profiles of Pseudomonas fluorescens: identification and properties.

The cell-associated peptidase profiles of 12 strains of Pseudomonas fluorescens (ATCC 948 and 11 related biotypes) were examined. Employing Analytab system API ZYM, a general, strong peptidase activity was detected using L-lysyl-, L-pyrrolidonyl-, L-arginyl-, L-alanyl-, and glycyl-glycyl-beta-naphthylamides as substrates. Conversely, L-tyrosyl-, L-phenylalanyl-, L-histidyl-, L-prolyl-, gamma-L-glutamyl-beta-naphthylamides substrates were hydrolyzed by only a few strains. The peptidases were active, therefore, on substrates responsible for the bitter taste in dairy products. Properties of hydrolytic systems showed no significant changes in the enzymatic profiles when cells were grown on different fermentation media. Enzyme activity was relatively stable during refrigerated (5 degrees C) and frozen (-18 degrees C) storage. The peptidases of P. fluorescens ATCC 948, considered as reference, and strain 22 were identified on Pro-beta-naphthylamides by Michaelis constant values of .528 and .394 mM, respectively, and by different optimal pH and temperature activity on Leu- and Pro-beta-naphthylamides. The peptidase activity on Gly-Phe-beta-naphthylamide in P. fluorescens 30 had optimal values at pH 7.50 and 45 degrees C. These results confirm the relations defined in the enzymatic identification phase and suggest the presence of any analogous peptidases in the biovars of P. fluorescens considered

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice

Free D- and L-Amino Acids from Hydrolyzed Milk Proteins by Pseudomonas fluorescens ATCC 948

Abstract Cell-associated peptidase activity of Pseudomonas fluorescens ATCC 948 was studied on hydrolyzed milk proteins. The substrate was produced by treatment of the UHT skim milk with neutral endoprotease B500. The cell-associated peptidase activity was determined by gas chromatographic analysis of free D- and L-amino acids. The total free amino acids were higher when the cell-associated peptidases acted on hydrolyzed milk proteins (202.8 μ g/ml) rather than on unhydrolyzed skim milk (63.9 μ g/ml). Glutamic acid (65.1 μ g/ml), Leu (36.9 μ g/ml), and Ala (16.5 μ g/ml) were the most abundant. Concentrations of D-amino acid isomers (28.3 and 3.7 μ g/d for D-Glu and D-Ala, respectively) also were high. Hydrolysis of the dipeptide L-leucyl-L-leu was 61% but was minimal for the other D- and L-configurational isomers of the dipeptide

Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds

Search for the standard model higgs boson in e+ e- collisions at s**(1/2) = 161-GeV - 172-GeV

This paper describes a search for the Standard Model Higgs boson using data from e^+e^- collisions collected at center-of-mass energies of 161, 170 and 172 GeV by the OPAL detector at LEP. The data collected at these energies correspond to integrated luminosities of 10.0, 1.0 and 9.4 pb^-1, respectively. The search is sensitive to the main final states from the process in which the Higgs boson is produced in association with a fermion anti-fermion pair, namely four jets, two jets with missing energy, and two jets produced together with a pair of electron, muon or tau leptons. One candidate event is observed, in agreement with the Standard Model background expectation. In combination with previous OPAL searches at center-of-mass energies close to the Z^0 resonance and the revised previous OPAL searches at 161 GeV, we derive a lower limit of 69.4 GeV for the mass of the Standard Model Higgs boson at the 95% confidence level.Comment: 33 pages, LaTeX, 12 eps figures included, submitted to Zeit. Phys.