Effect of the oral intake of yogurt containing Bifidobacterium longum BB536 on the cell numbers of enterotoxigenic Bacteroides fragilis in microbiota

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been suggested to be associated with acute and persistent diarrheal disease, inflammatory bowel disease and colorectal cancer, although further epidemiological studies are needed for clarification. Here, a pilot study was performed to examine the effect of the oral administration of yogurt supplemented with a probiotic strain on the cell numbers of fecal ETBF in a healthy population. Among 420 healthy adults, 38 subjects were found to be ETBF carriers, giving a prevalence of approximately 9%. Among them, 32 subjects were enrolled in an open, randomized, parallel-group study to ingest yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536 (BB536Y group), for 8 weeks, with milk provided to the control group (milk group). The cell numbers of ETBF and the dominant species of the B. fragilis group were measured by a quantitative PCR method. Compared with the baseline values, there was a significant decrease in the cell number of ETBF at week 8 in the BB536Y group but not in the milk group. Linear mixed models analysis for longitudinal data revealed a significant difference in the changes of ETBF cell number between the two groups during the intervention phase. These results imply the potential of probiotic yogurt for eliminating ETBF in the microbiota, but its clinical significance needs to be evaluated in the future. This is the first report of a possible effect of probiotic intake on ETBF in the microbiota.     

Suppressive effects of Bifidobacterium longum on the production of Th2-attracting chemokines induced with T cell–antigen-presenting cell interactions

In human trials, Bifidobacterium longum BB536 alleviates subjective symptoms of Japanese cedar pollinosis, an IgE-mediated type I allergy caused by exposure to Japanese cedar, and significantly suppresses the increase of plasma thymus- and activation-regulated chemokine (TARC) associated with pollen dispersion. In the present study, we investigated the suppressive effects of BB536 on the production of T helper type 2 (Th2)-attracting chemokines, such as TARC and macrophage-derived chemokine (MDC), together with the mechanisms of their production. Murine splenocytes were cultured with heat-killed BB536, and the levels of Th2-attracting chemokines in the supernatants were measured. TARC and MDC were produced in cultures without stimulation, and the production was significantly suppressed by BB536. These chemokines were produced by antigen-presenting cells (APCs) of splenocytes stimulated with an anti-CD40 antibody. Furthermore, TARC production was induced with granulocyte macrophage colony-stimulating factor that was produced by T cells and dendritic cells. BB536 suppressed MDC production induced with the anti-CD40 antibody by APCs from the spleen, mesenteric lymph nodes (MLNs) and Peyer's patches, and it suppressed TARC production by APCs from the spleen and MLNs. These results indicate that BB536 suppresses the production of Th2-attracting chemokines induced by the T cell–APC interaction, suggesting a novel mechanism for alleviating symptoms of allergic disorders by probiotics.     

Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L33180","term_id":"3745835","term_text":"L33180"}}L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

Nucleopolyhedroviruses (NPVs) have large (80- to 180-kb) circular double-stranded DNA (dsDNA) genomes, which replicate in nuclei of infected cells. Despite the widespread use of NPVs for the expression of foreign genes and their potential for pest control, little is known about the mechanism of their replication and the properties of their replication factors. The most widely studied baculovirus, Autographa californica multicapsid NPV (AcMNPV), has the potential to encode about 150 proteins (3), including factors required for virus DNA replication. The products of nine viral genes (ie-1, ie-2, lef-1, lef-2, lef-3, dnahel, dnapol, p35, and lef-7 or pe-38) are necessary and sufficient for efficient replication of transfected plasmid DNAs containing a putative baculovirus replication origin (16, 22). It is likely that DNA polymerase and DNA helicase, which are encoded by the viral genes dnapol and dnahel, respectively (20, 35), form a core of the virus DNA replication machinery. The roles of other factors are less obvious. Single-stranded DNA binding (SSB) protein function was proposed for the protein LEF-3, which binds specifically single-stranded DNA (ssDNA) (10, 14). However, direct proof for the SSB function of LEF-3 in viral DNA replication is lacking. In addition, SSB function was also suggested for LEF-7 on the basis of its predicted amino acid sequence (22). It was recently demonstrated that LEF-1 forms a complex with LEF-2 and may serve as a DNA primase (9). The function of IE-1, IE-2, and PE-38 may result from their ability to activate in trans expression of other genes required for virus replication. The transactivator IE-1 may also participate in the initiation of DNA replication, due to its ability to bind putative replication origins (7, 13, 17, 33). P35 is an inhibitor of apoptosis and may not be involved directly in DNA replication. Its stimulatory effect in the transient-replication assay may result from inhibition of virus-induced apoptosis in cells transfected with the replication genes. Several genes required for DNA replication (six essential and three stimulatory) were also identified in the genome of Orgyia pseudotsugata NPV (1). Homology of these genes to those required for replication of AcMNPV suggests similar replication mechanisms for the two viruses. The genome organization of the Bombyx mori NPV (BmNPV) closely resembles that of AcMNPV. Nineteen homologs of the AcMNPV late expression factor genes (lef genes) were identified in BmNPV (12). At least three of these, ie-2, lef7, and p35, are not essential for virus DNA replication as demonstrated by deletion analysis (12). Because the daughter DNA molecules synthesized under control of the nine essential viral genes appear to be synthesized as concatemers (16, 22, 31, 32), factors required for maturation of nascent DNA and its further processing are still unknown. Although the nine AcMNPV factors were sufficient for efficient DNA replication in Sf cells, an additional viral gene, designated hcf-1, was essential for replication in TN-368 cells (21), indicating dependence of the transient assay on host cell-specific factors. Few proteins involved in NPV DNA replication have been purified from infected cells and characterized in cell-free systems. Among them are AcMNPV DNA polymerase (28, 37), BmNPV DNA polymerase (27), AcMNPV DNA helicase (19), and AcMNPV LEF-3 (10, 14). Isolation of other replication proteins of NPVs is still anticipated.In this report we describe the purification of a viral DNA-binding protein (designated DBP) from BmNPV-infected cells. DBP binds preferentially to ssDNA and is capable of unwinding duplex DNA. The BmNPV open reading frame (ORF) encoding DBP (dbp gene) is a homolog of AcMNPV ORF25, whose product has not been identified so far.     

Hydroxyl radical and singlet oxygen production and DNA damage induced by carcinogenic metal compounds and hydrogen peroxide

Carcinogenic chromium(VI), iron(III) nitrilotriacetate, cobalt(II), and nickel(II) react with hydrogen peroxide leading to the production of active species including hydroxyl radical and singlet oxygen, which cause DNA damage.     

Patterns of Protein Production in Myxococcus xanthus During Spore Formation Induced by Glycerol, Dimethyl Sulfoxide, and Phenethyl Alcohol

Spore formation of Myxococcus xanthus can occur not only on agar plates during fruiting body formation, but also in a liquid culture by simply adding glycerol, dimethyl sulfoxide, or phenethyl alcohol to the culture. This chemically-induced spore formation occurs synchronously and much faster than that occurring during fruiting body formation. Dramatic changes in patterns of protein synthesis were observed during chemically-induced spore formation, as had previously been observed during fruiting body formation (Inouye et al., Dev. Biol. 68:579-591, 1979). However, the production of protein S, one of the major development-specific proteins during fruiting body formation, was not detected at all, although protein U, another development-specific protein, was produced in a late stage of spore formation as in the case of fruiting body formation. This indicates that the control of the gene expression during chemically-induced spore formation is significantly different from that during fruiting body formation. It was also found that during spore formation, every cell seems to have a potential to form a spore regardless of its age, since smaller cells as well as larger cells separated by sucrose density gradient centrifugation could equally form spores upon the addition of glycerol. Patterns of protein synthesis were almost identical for all the three chemicals. However, the final yield of spores was significantly different depending upon the chemicals used. When phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals added. This suggests that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation.     

Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA

In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.     

Inhibition of Development of Myxococcus xanthus by Eukaryotic Protein Kinase Inhibitors

Myxococcus xanthus is a social bacterium that lives in the soil and undergoes spectacular development to form multicellular fruiting bodies. It contains a large family of eukaryote-like serine/threonine protein kinases. We found that a number of inhibitors for eukaryotic protein serine, threonine, and tyrosine kinases could inhibit the development and sporulation of M. xanthus to various degrees. These results suggest that serine/threonine and tyrosine phosphorylation may be involved in development of M. xanthus. None of the inhibitors tested had any effect on vegetative growth of M. xanthus. Most of them seemed to act during the early stages of development. However, the expression of a very early development-specific gene, Ω4521, was not significantly affected by the inhibitors. The patterns of protein phosphorylation during development were also not significantly altered by the inhibitors, suggesting that the targets of the inhibitors are minor or unstable phosphoproteins but play key roles in fruiting-body formation in M. xanthus.