A novel multifunctional peptide oligomer of bacitracin with possible bioindustrial and therapeutic applications from a Korean food-source <i>Bacillus</i> strain - Fig 1

<p>(A) A neighbor-joining tree based on nearly complete 16S rRNA gene sequences showing relations between strain CS32 and related members of the genus <i>Bacillus</i>. The percentages at the nodes are the levels of bootstrap support based on neighbor-joining analyses of 1000 resampled datasets. The sequence of <i>Bacillus licheniformis</i> ATCC 14580(T) served as an outgroup. The scale bar: 0.01 nucleotide substitutions per position. (B) Production of CSP32 and the growth-inhibitory activity against <i>Mycobacterium smegmatis</i> ATCC 9341. The antibacterial activity is maximal at 60 h. The zone of inhibition against <i>Mycobacterium smegmatis</i> ATCC 9341 at every 12 h is shown. Cultivation was carried out in 250-mL flasks with 50 mL of a medium, at pH 7 and 37°C, with shaking at 180 rpm.</p

Effects of CSP32 on NO production and expression of inflammation-associated proteins in LPS-stimulated RAW 264.7 macrophage cells.

<p>The cells were treated with LPS (1 μg/mL) in the presence of various concentrations of CSP32 (10, 50, or 100 μg/mL) at 37°C for 24 h. (A) The amounts of NO were determined using the Griess reagent in the culture medium (Top panel). Equal amounts of cell lysates were resolved on SDS polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against iNOS and COX-2. β-Actin served as an internal control for western blot analysis (bottom panel). Effects of CSP32 on LPS-induced mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6. (B) After LPS treatment for 2–12 h, the levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR. <i>GAPDH</i> served as an internal control for RT-PCR assays. Suppressive effects of CSP32 on TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 macrophage cells. (C) The cells were incubated with the indicated concentrations of CSP32 for 30 min before treatment with LPS (1 μg/mL) for 24 h. After incubation for 24 h, the supernatant was collected, and the amounts of these proinflammatory cytokines were measured by ELISAs. Results represent the mean±S.D of three independent experiments performed in triplicate. * p<0.01; compared to LPS alone.</p

Comparison of MS data with predicted molecular weight of each subunit.

<p>Comparison of MS data with predicted molecular weight of each subunit.</p

Comparative analysis of N-terminal amino acid sequences.

<p>Comparative analysis of N-terminal amino acid sequences.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p