Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter

Suppression of LFA-1 expression by spermine is associated with enhanced methylation of ITGAL, the LFA-1 promoter area.

Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1) expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO) significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt) activity, enhanced demethylation of the LFA-1 gene (ITGAL) promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM) of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM), and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM-such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase) and SAM supplementation-successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL

Changes in methylation status of ITGAL promoter by DFMO and spermine supplementation.

<p>Bisulfite sequencing was performed to determine the methylation pattern of the Jurkat cell ITGAL promoter, numbered relative to the transcription start site. Left: methylation status of each CpG dimer in 4 segments of sequential fragments. Each line: 1 experiment. Number of lines: number of experiments. Black circle: methylated CpG dimers. White circle: demethylated CpG dimers. Right upper: effects of DFMO on methylation status. Right lower: methylation status changes after spermine supplementation of cells treated with DFMO. Percentage: the increase of methylated CpG dimer; positive values, increased methylation; negative values, increased demethylation. ITGAL: LFA-1 gene, DFMO: D,L-alpha-difluoromethylornithine hydrochloride.</p

Effect of DFMO and spermine supplementation on Active Rap-1 protein levels.

<p>Neither DFMO nor spermine activated Rap-1. negative control: cell lysate treated with GDP. positive control: cell lysate treated with GTP-gamma-S. no treatment: cell lysate with no treatment. DFMO: lysate of cells cultured with 3 mM DMFO. DFMO+spermine: lysate of cells cultured with 3 mM DMFO and 500 µM spermine. Rap-1: Ras-proximate-1, DFMO: D,L-alpha-difluoromethylornithine hydrochloride.</p

Effect of interventions affecting polyamine metabolism on CD11a expression.

<p>The mean fluorescent intensities of CD11a in Jurkat cells cultured for 72 h in various conditions were analyzed by flow cytometry. Mean ± standard deviation; n = number of experiments. control: cells cultured in unsupplemented culture medium. DFMO: cells cultured in control medium plus 3 mM DMFO. DFMO+spermine: cells cultured in control medium plus 3 mM DMFO and 500 µM spermine. DFMO+MGBG: cells cultured in control medium plus 3 mM DMFO and 0.25 µM MGBG. DFMO+SAM: cells cultured in control medium plus 3 mM DMFO and 50 µM SAM. DFMO: D,L-alpha-difluoromethylornithine hydrochloride, MGBG: methylglyoxal bis-guanylhydrazone, SAM: S-adenosylmethionine.</p

Effect of DFMO and spermine supplementation on Dnmt activity.

<p>DFMO decreased, while spermine supplementation increased Dnmt activity in Jurkat cells. Mean ±standard deviation; n = number of experiments as indicated. no treatment (control): cells cultured in culture medium. DFMO: cells cultured with 3 mM DMFO. DFMO+spermine: cells cultured with 3 mM DMFO and 500 µM spermine. DFMO: D,L-alpha-difluoromethylornithine hydrochloride.</p

Primers used for bisulfite sequencing (secondary PCR).

<p>Primers used for bisulfite sequencing (secondary PCR).</p