Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis

Summary HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented. This research was supported by Oklahoma Agricultural Experiment Station Project 1534. This publication is Article J-3334 of the Oklahoma Agricultural Experimental Station.     

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.     

Histamine enhances cytotrophoblast invasion by inducing intracellular calcium transients through the histamine type-1 receptor

Blastocyst implantation and placentation require molecular and cellular interactions between the uterine endometrium and blastocyst trophectoderm. Previous studies showed that histamine produced in the mouse uterine luminal epithelium interacts with trophoblast histamine type-2 receptors (H2) to initiate blastocyst implantation. However, it is unknown whether similar histamine activity is operative in humans. Using a human cell line (HTR-8/SVneo) derived from first-trimester cytotrophoblasts that expresses both histamine type-1 receptor (H1) and H2, we found that histamine promotes cytotrophoblast invasiveness specifically through activation of H1. Stimulation of H1 in human cytotrophoblasts by histamine induced intracellular Ca2+ (Ca(2+)i) transients by activating phospholipase C and the inositol trisphosphate pathway. The enhanced invasion induced by histamine was blocked by pretreatment with H1 antagonist or by chelation of Ca(2+)i. These findings suggest possible differences between rodents and humans in histamine signaling to the trophoblast.     

Bioinformatic analyses of the tRNA: (guanine 26, N2,N2)-dimethyltransferase (Trm1) family

Functional analyses of the tRNA:(guanine 26, N2,N2)-dimethyltransferase (Trm1) have been hampered by a lack of structural information about the enzyme and by low sequence similarity to better studied methyltransferases. Here we used computational methods to detect novel homologs of Trm1, infer the evolutionary relationships of the family, and predict the structure of the Trm1 methyltransferase. The N-terminal region of the protein is predicted to form an S-adenosylmethionine-binding domain, which harbors the active site. The C-terminal region is rich in predicted alpha-helices and, in analogy to other nucleic acid methyltransferases, may constitute the target recognition domain of the enzyme. Interposing these two domains, most Trm1 homologs possess a highly variable inserted sequence that is delimited by a Cys4 cluster, likely forming a Zn-finger structure. The residues of Trm1 predicted to participate in cofactor binding, target recognition, and catalysis, were mapped onto a preliminary structural model, providing a platform for designing new experiments to better understand the molecular functions of this protein family. In addition, identification of novel, atypical Trm1 homologs suggests candidates for cloning and biochemical characterization.     

Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin-saccharide interactions

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.     

Extended Survival and Persistence of Campylobacter spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining

In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.     

Distribution of Xanthomonas oryzae pv. oryzae DNA Modification Systems in Asia

The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI⁺ XorII⁺, XorI⁺ XorII⁻, XorI⁻ XorII⁺ and XorI⁻ XorII⁻) were detected in the population at a ratio of approximately 1:2:2:2. The XorI⁺ XorII⁺ and XorI⁻ XorII⁺ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI⁻ XorII⁻ and XorI⁺ XorII⁻ were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII⁺ strains within a country, indicating clonal maintenance of the XorII methyltransferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.     

Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB

The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.