Temperature-hypersensitive sites of the flagellar switch component FliG in Salmonella enterica serovar typhimurium

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla(-), Mot(-), and Che(-)), depending on the sites and types of mutations. Some of the Mot(-) mutants previously characterized are found to be motile. Reexamination of all Mot(-) mutants in fliGMN genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20 degrees C but nonmotile at 37 degrees C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of fliM and fliN and most of the fliG TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the fliG mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.     

Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m⁻² s⁻¹ with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.     

Antagonist of secondary lymphoid-tissue chemokine (CCR ligand 21) prevents the development of chronic graft-versus-host disease in mice

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient's lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 x DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.     

Pharmacokinetic Study of Adjuvant Gemcitabine Therapy for Biliary Tract Cancer following Major Hepatectomy (KHBO1101)

Background

Biliary tract cancer (BTC) patients who have undergone surgical resection with major hepatectomy cannot tolerate the standard gemcitabine regimen (1,000 mg/m² on days 1, 8, and 15 every 4 weeks) due to severe toxicities such as myelosuppression. Our dose-finding study of adjuvant gemcitabine therapy for biliary tract cancer following major hepatectomy determined that the recommended dose is 1,000 mg/m² on days 1 and 15 every 4 weeks. Here, we evaluate the pharmacokinetics and pharmacodynamics of gemcitabine in these subjects.

Methods

We evaluated BTC patients scheduled to undergo surgical resection with major hepatectomy followed by gemcitabine therapy. A pharmacokinetic evaluation of gemcitabine and its main metabolite, 2′,2′-difluorodeoxyuridine (dFdU), was conducted at the initial administration of gemcitabine, which was given by intravenous infusion over 30 min at a dose of 800–1,000 mg/m². Physical examination and adverse events were monitored for 12 weeks.

Results

Thirteen patients were enrolled from August 2011 to January 2013, with 12 ultimately completing the pharmacokinetic study. Eight patients had hilar cholangiocarcinoma, three had intrahepatic cholangiocarcinoma, and one had superficial spreading type cholangiocarcinoma. The median interval from surgery to first administration of gemcitabine was 65.5 days (range, 43–83 days). We observed the following toxicities: neutropenia (n = 11, 91.7%), leukopenia (n = 10, 83.3%), thrombocytopenia (n = 6, 50.0%), and infection (n = 5, 41.7%). Grade 3 or 4 neutropenia was observed in 25% (n = 3) of patients. There were differences in clearance of gemcitabine and dFdU between our subjects and the subjects who had not undergone hepatectomy.

Conclusion

Major hepatectomy did not affect the pharmacokinetics of gemcitabine or dFdU.

Trial Registration

UMIN-CTR in (JPRN) UMIN000005109     

Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.     

Azalide 3,6-ketals: antibacterial activity and structure-activity relationships of aryl and hetero aryl substituted analogues

Aryl and hetero aryl substituted 3,6-ketals of 15-membered azalide analogues were synthesized and were found to have potent in vitro antibacterial activity against veterinary pathogens, including Staphylococcus aureus and Pasteurella multocida.     

Shortening of human cell life span by induction of p16ink4a through the platelet-derived growth factor receptor β

Mesenchymal stem cells (MSCs) are attracting a great deal of attention because they represent a valuable source of cells for use in regenerative medicine. In human cell culture it is important to obtain large numbers of cells for use in therapy. In this study, we attempted to prolong life span of a marrow-derived mesenchymal stem cell using a combination of growth factors and hormones. Furthermore we tested whether chemically defined culture conditions are sufficient for maintenance of multipotent mesenchymal stem cells. Epidermal growth factor, platelet-derived growth factor-BB (PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF, and leukemia inhibitory factor were found to be key factors for the mesenchymal stem cell proliferation. The combination of these growth factors showed extremely strong mitogenic activity, and simultaneously induced the expression of cyclin-dependent kinase inhibitor p16ink4a protein and premature senescence more rapidly than serum-supported culture conditions. The induction of p16ink4a by growth factors was mediated through the mitogen-activated protein kinase (MAPK) cascade. Excess growth stimulation by growth factors was thus one of the culture stress signals and a trigger of premature senescence at least in human cells. J. Cell. Physiol. 221: 335–342, 2009. © 2009 Wiley-Liss, Inc.     

Decreased serum leptin and muscle oxidative enzyme activity with a dietary loss of intra-abdominal fat in rats

The purpose of the present study was to investigate the relationship among intra-abdominal adipose storage, adaptation in the serum leptin concentration and skeletal muscle enzyme activity after a 4-week energy restriction (ER). Thirty-one male Wistar rats were divided into 40% energy restricted (n=24) or ad libitum-fed control (CL) rats (n=7). The energy-restricted rats were grouped into the most fat (MF, n=7), medium (n 10) and the least fat (LF, n=7) by their intra-abdominal fat pads mass (epididymal, mesenteric, and perirenal) after ER. A superficial portion of M. gastrocnemius tissue obtained before and after the diet period were analyzed to determine the activities of hexokinase (HK), beta-hydroxyacyl CoA dehydrogenase (beta-HAD) and citrate synthase (CS). Blood samples were also collected for a serum leptin assay. At the baseline, no difference was found in either the leptin concentration or the enzyme activities among LF, MF and CL. The serum leptin concentration was positively correlated with the muscle activities of beta-HAD and CS, while it negatively correlated with HK/beta-HAD. After ER, the activities of HK, beta-HAD and CS were all significantly lower in LF than in CL. Among the energy-restricted rats, the intra-abdominal fat pad weight, leptin concentration and the activities of beta-HAD, CS, beta-HAD/CS all significantly correlated with one another. The changes in leptin and the activity of beta-HAD were also positively correlated. These findings indicate that parallel decreases in the serum leptin and skeletal muscle enzyme activities with the energy restriction-induced intra-abdominal adipose reduction, thus may suggest the leptin to have a regulative effect on the muscle enzyme activity during ER.