Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing

Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater. Received: 19 May 1998; Accepted: 29 October 1998     

Stoichiometric and catalytic hydrogenation of the [Co2(CO)6(μ2-η2-alkyne)] complexes

The [Co₂(CO)₆(RC₂R′)] complexes (R, R′ = H, Me, Et, Prⁿ) react with molecular hydrogen under mild conditions of temperature and pressure, at low but appreciable rates. The effect of the steric hindrance of the substituents and the strength of the metalcarbon bonds are discussed. The kinetic data measured for [Co₂(CO)₆(HC₂H)], suggest that both H₂-coordination and CO-dissociation are involved in the rate-determining step of the overall hydrogenation process.The catalytic activity of [Co₂(CO)₆(HC₂H)] in the homogeneous hydrogenation of acetylene is described. At low substrate/catalyst ratio the initial hydrogenation rate is equal, within experimental error, to that found for the stoichiometric reaction; on increasing the acetylene concentration, cyclotrimerization to benzene becomes the dominant process. Interestingly C₄ hydrocarbons (mainly butadiene and 1-butene) are produced in measurable yield (?8%). The formation of these products is interpreted as the result of the hydrogenation of the elusive [Co₂(CO)₅(HC₂H)₂] complex, an unstable intermediate in the cyclotrimerization chain.     

A preliminary report on the use of transfer factor for treating stage D3 hormone-unresponsive metastatic prostate cancer

As conventional treatments are unsuccessful, the survival rate of stage D3 prostate cancer patients is poor. Reports have suggested the existence of humoral and cell-mediated immunity (CMI) against prostate cancer tumour-associated antigens (TAA). These observations prompted us to treat stage D3 prostate cancer patients with an in vitro produced transfer factor (TF) able to transfer, in vitro and in vivo, CMI against bladder and prostate TAA. Fifty patients entered this study and received one intramuscular injection of 2–5 units of specific TF monthly. Follow-up, ranging from 1 to 9 years, showed that complete remission was achieved in 2 patients, partial remission in 6, and no progression of metastatic disease in 14. The median survival was 126 weeks, higher than the survival rates reported in the literature for patients of the same stage.     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

Ethanol inhibits ligand-activated Ca2+ channels in human B lymphocytes.

Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B-cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo-1 following stimulation with either anti-IgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto-oncogene c-fos in these cells. Ethanol inhibited ligand-activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose- and time-dependent manner. This inhibition was not due to the inability of anti-IgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2(+)-dependent induction by anti-IgM of c-fos in these cells. The inhibitory effects of ethanol on ligand-activated Ca2+ channels and subsequent induction of c-fos may provide the basis for its immunosuppressive action.     

Genetic variation and drought response in two Populus × euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides × Populus nigra, cv. ‘Agathe_F’ and ‘Cima’, were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra.A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, ‘Cima’ displayed the highest abundance of antioxidant enzymes.In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while ‘Cima’ differed from ‘Agathe_F’ by increased abundance of enzymes involved in photorespiration and in oxidative stress, ‘Agathe_F’ was mainly differentiated by increased abundance of enzymes involved in photosynthesis.