Detection and changes of nucleotide pyrophosphotransferase activity of Streptomyces griseus strains in submerged culture.

Nucleotide pyrophosphotransferase (NPT) activity of two Streptomyces griseus strains was studied in submerged culture during their life cycle. NPT activity could be detected only in the culture filtrate but not in the membrane fraction or in cell extract of the sporulating (streptomycin-non-producing) S. griseus No. 45-H. No enzyme could be detected in the non-sporulating (streptomycin-producing) S. griseus No 52--1 cultures.     

Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination

Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development.     

Origin and fixation of pyrophosphates in the parietal bones of young rats]

Calvaria from 6 to 13-day-old rats mineralize and bind pyrophosphates which are transferred from nucleotides, as shown by : 1 -- the increase of pyrophosphates in young rat calvaria incubated with nucleoside triphosphate, especially ATP ; 2 -- the more important increase of pyrophosphate content when ATP is renewed in the incubation medium ; and 3 -- binding of [32P] pyrophosphate [beta 32P] ATP. The effect of preheating of calvaria on 32P binding from [beta32P]ATP led us to assume that two systems might be involved in pyrophosphate fixation : a heat-labile, non-specific, phosphatase system, and a heat-stable pyrophosphotransferase system. cAMP increases the pyrophosphate content of calvaria incubated with ATP : that effect may result from either an inhibition of the phosphatase system, or an activation of the pyrophosphotransferase system.     

GTP-specific pyrophosphorylation of thiamin in dark-grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900-fold from 48 h dark-grown soybean [ Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2-¹⁴C]-thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent K_ms from 21 to 138 m M ), GTP was the preferred nucleotide with an apparent K_m of 0.021 m M . TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H⁺ and Mg²⁺ concentrations, either independently or in concert, were found to affect TPT activity.     

Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings

Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2-¹⁴C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent K_m values of 18.3 millimolar and 4.64 micromolar were obtained for Mg·ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000.     

Purification and Some Properties of ATP: Nucleotide Pyrophospho-transferase of Streptomyces adephospholyticus

ATP: nucleotide pyrophosphotransferase was purified from culture filtrate of Streptomyces adephospholyticus A–4668 about 13,000 fold by the method including ammonium sulfate fractionation, Amberlite IRC–50 treatment and column chromatography with DEAE-cellulose, DEAE-Sephadex A–25, SP-Sephadex C–25 and Sephadex G–75. The purified enzyme was homogenous on disk gel electrophoresis and ultracentrifugation and the specific activity was 915 units per mg protein, The molecular weight was determined as 28,000 by gel filtration on Sephadex G–75. The enzyme was found to be stable in the pH range of 5.5 to 10.5. More than 80% of the activity was remained after heating at 60°C for 30 min. The enzyme exhibited maximum activity at 50°C.     

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.     

Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin

Among 828 C. diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains. All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin. The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C. diphtheriae toxigenic strains. The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon. The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C. diphtheriae nontoxigenic strains is spread in Russia. These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.     

Isolation and Identification of ATP: Nucleotide Pyrophospho-transferase-producing Microorganism

ATP: nucleotide pyrophosphotransferase-producing microorganism was isolated from soil in Osaka prefecture. The morphological and physiological characteristics of this microorganism were studied. This strain was identified and named Streptomyces adephospholyticus nov. sp.

When this strain was aerobically cultured in a fermentor at 30°C in a medium containing 2% glycerol, 4% polypepton, 0.1 % KH₂PO₄, 0.04% MgSO₄ · 7H₂O, 2 ppm FeSO₄ · 7H₂O and 2 ppm MnSO₄ · 6Н₂О at pH 7.0, ATP; nucleotide pyrophosphotransferase was produced in the culture filtrate. The highest activity was obtained after 30 to 40 hr cultivation. The maximum enzyme production was 3000 to 4000 unit per liter.     

Nucleotide composition and homologies in the DNA of new extreme halophilic soil archaebacteria

DNA nucleotide composition was studied in extreme halophilic bacteria belonging to the genera Halobacterium, Halococcus, Natronobacterium and Natronococcus. The cultures were shown to be a monolithic group of microorganisms with the content of GC pairs typical of extreme halophilic archebacteria. The difference between the content of DNA major and minor components was twice as high in Halobacterium distributus strains isolated from sulfate saline soils as compared to cultures of this species isolated from natural waters with a high salinity. DNA minor components were not found in haloalkalophilic microorganisms from soda saline soils in contrast to those from soda lakes. The results of DNA-DNA hybridization indicate that the Halobacterium genus is highly heterogeneous. The newly isolated strains of extremely halophilic H. distributus are characterized by the low homology of their DNAs both among themselves and with other species of the genus. However, the hybridization data for the collection strains H. vallismortis 1398 and H. halobium 996 from the National Collection of Microorganisms are indicative of a high homology (80-100%) which is not characteristic of cultures belonging to different species. These results as well as some phenotypical properties of H. vallismortis 1398 different from those of this species type strain support the data reported in the literature about the genetic instability of extreme halophilic archebacteria. The analysis of homologies in DNA nucleotide sequences may be used to study the taxonomy of extreme halophilic archebacteria.     

Revised classification of native probiotic strains of Lactobacillus used in Russian Federation

Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).     

Evidence that measles virus hemagglutinin initiates modulation of leukocyte function-associated antigen 1 expression.

Measles virus (MV), human immunodeficiency virus, Epstein-Barr virus, and other leukotropic viruses can modulate the expression of leukocyte function antigen 1 (LFA-1) on the surface of infected and nearby leukocytes. This ability to induce changes in LFA-1 expression may play an important role in the pathogenesis of these viruses. However, the mechanism(s) involved in virus-mediated regulation of LFA-1 is unknown. Evidence is presented in this report that it is the MV hemagglutinin (H) protein that initiates up-regulation of LFA-1 expression in leukocyte cultures infected with this virus. Indeed, comparison of the abilities of different MV strains to modulate LFA-1 expression, examination of published nucleotide sequences for the H proteins of different vaccine strains, and competitive inhibition assays using oligopeptides homologous or heterologous to a region of the H protein gene encompassing amino acid 116 (from the amino terminus) all suggest that it is this portion of the H protein that is responsible for MV-induced alteration of LFA-1. These comparisons also support the hypothesis that there is a relationship between the abilities of different MV strains to alter LFA-1 expression and their pathogenic potentials.     

Acceptor specificity of ATP:nucleoside-5'-phosphate pyrophosphotransferase from Streptomyces adephospholyticus. Synthesis of the 3'-pyrophosphates of pyrimidine nucleotides, some oligoribonucleotides, 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am.

ATP: nucleoside-5'-phosphate pyrophosphotransferase [EC 2.7.6.4] of Streptomyces adephospholyticus synthesizes not only 3'-pyrophosphates of 5'-purine ribomononucleotides but also those of pyrimidine mononucleotides, some short oligonucleotides, a variety of 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am.     

Plasmid profiles of Lactobacillus plantarum spp. found as contaminants in Hemerocallis plant tissue cultures

Plasmid profiles of 30 strains of Lactobacillus plantarum isolated from 3-year-old tissue cultures and surface sterilized stem sections of glasshouse grown plants of three different varieties of Hemerocallis were compared to pinpoint the source of Lact. plantarum contamination in plant tissue cultures. Since the profiles of strains isolated from in vitro and in vivo plant material were generally identical, it is thought that the source of Lact. plantarum contamination is the plant material used to initiate plant tissue cultures.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.     

Genetic characteristics of the KC vaccine strain of hog cholera virus: comparative analysis of the primary sequence of surface glycoprotein E(rns), E1, and E2 genes]

Primary structure of a genome fragment of attenuated strain CS of hog cholera virus (HCV) coding for three surface glycoproteins Erns, E1, and E2 (fragment size 2379 nucleotides) is analyzed. By the nucleotide sequence the homology between strain CS and ten other virulent and attenuated HCV strains in this area is 84.9-94.6%, 87.2-94.6% in gene Erns, 84.6-96.9% in gene E1, and 83.3-94.3% in gene E2. By amino acid sequence the homology is 90.9-94.3%, 92.9-95.0%, 92.3-95.6%, and 88.9-94.1%, respectively. Computer analysis demonstrated philogenetic ratios between these strains and other HCV strains and the areas of potential antigenic differences between CS strain and other HCV strains. The data indicate that strain CS used as live vaccine protecting from HCV contains unique nucleotide and amino acid positions and its evolution history is different from that of analyzed reference strains. The data will be further used for detecting the fine antigenic structure of strain CS surface glycoproteins with the aim of disclosing unique antigenic markers.     

Antibiotic sensitivity of staphylococci isolated from the surface of the skin of the breasts in pregnant women

Nine hundred and fourty coagulase-positive and coagulase-negative strains of staphylococci isolated from the skin surface of the mammary glands of 94 pregnant women were tested by the disc agar diffusion method for their sensitivity to five antibiotics. The highest number of the isolates were sensitive to erythromycin and lincomycin (87.7 and 89.7 per cent, respectively). The highest number of moderately resistant strains were detected with respect to methicillin. 19 out of 42 cultures of Staph. aureus were resistant to benzylpenicillin and 24 cultures were resistant to tetracycline. Among staphylococci 130 strains or 13.8 per cent were polyresistant.