A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Inhibitory action of a macrolide antibiotic,roxithromycin, on co-stimulatory molecule expressions in vitro and in vivo

OBJECTIVE: The influence of a macrolide antibiotic, roxithromycin (RXM), on co-stimulatory molecule expression was examined in vitro and in vivo. MATERIALS AND METHODS: Spleen cells obtained from BALB/c mice 10 days after immunization with 8.0 microg of hemocyanin absorbed to 4.0 mg of aluminum hydroxide were cultured in the presence of 100.0 microg/ml of hemocyanin and various concentrations of RXM. We first examined the influence of RXM on cell activation by examining the proliferative response of cells and cytokine production. We also examined the influence of RXM on co-stimulatory molecule (CD40, CD80 and CD86) expressions on cultured splenic B-lymphocytes induced by in vitro antigenic stimulation using flow cytometry. In the second part of experiments, non-immunized and immunized mice were treated orally with 2.5 mg/kg of RXM once a day for 4 or 8 weeks. Splenic B lymphocytes were obtained from these mice 24 h after antigenic challenge, and co-stimulatory molecule expressions were examined by flow cytometer. RESULTS: Cell activation induced by in vitro antigenic stimulation was suppressed by RXM when cells were cultured in the presence of more than 5.0 microg/ml of the agent. Addition of RXM at a concentration of 5.0 microg/ml into cell cultures also suppressed co-stimulatory molecule (CD40, CD80 and CD86) expressions on splenic B lymphocytes, which was enhanced by antigenic stimulation in vitro. Oral RXM administration for 4 weeks clearly suppressed the enhancement of CD40 and CD86 (but not CD80) expressions on splenic B lymphocytes induced by antigenic stimulation in vivo. This suppressive activity of RXM on co-stimulatory molecule (CD40 and CD86) expressions was further strengthened by the treatment of mice for 8 weeks. Long-term treatment with oral RXM also suppressed CD80 expressions, which was not suppressed by 4-week treatment. CONCLUSION: The present results suggest that RXM exerts its immunomodulating effects through suppression of both cell activation and co-stimulatory molecule expressions induced by antigenic stimulation. These suppressive activities of RXM might contribute, in part, to the therapeutic mode of action of RXM on inflammatory diseases.     

tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria

The 2,4-dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdAalpha was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the alpha subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAalpha to representative tfdA genes. A MalE-TfdAalpha fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an alpha-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAalpha revealed a conserved His-X-Asp-X(146)-His-X(14)-Arg motif characteristic of the active site of group II alpha-ketoglutarate-dependent dioxygenases. The tfdAalpha genes were also detected in 2,4-D-degrading alpha-Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63:2266-2272, 1997). These findings indicate that the tfdA genes in beta- and gamma-Proteobacteria and the tfdAalpha genes in alpha-Proteobacteria arose by divergent evolution from a common ancestor.     

Imprint cytology of epithelioid angiomyolipoma in a patient with tuberous sclerosis. A case report

BACKGROUND: Angiomyolipoma composed predominantly of epithelioid cells has been referred to as epithelioid angiomyolipoma. As this subtype shows considerable cellular atypia, it may be erroneously diagnosed as malignant epithelioid tumor, such as renal cell carcinoma and hepatocellular carcinoma. So far, only one report describing the cytologic findings of epithelioid angiomyolipoma has been documented, and epithelioid angiomyolipoma occurring in the peritoneal cavity has not been reported. CASE: Eleven years after resection of a renal epithelioid angiomyolipoma in a 34-year-old male with tuberous sclerosis, a tumor appeared in the peritoneal cavity and three masses in the liver. The intraoperative smears imprinted from part of the peritoneal mass revealed many large, atypical cells. The well-preserved atypical cells showed abundant, round to polyhedral, granular cytoplasm. Bizarre, giant nuclei with hyperchromasia and huge nucleoli were occasionally seen. Intranuclear cytoplasmic inclusions and mitotic figures were occasionally observed. As the epithelioid cells were markedly pleomorphic, we could not rule out hepatocellular carcinoma, cytologically and histologically, in the intraoperative consultation. In permanent sections the tumor was composed predominantly of epithelioid cells showing an alveolar pattern or sheetlike arrangement. Mitotic counts were zero to one per 10 high-power fields. Immunohistochemically, the epithelioid tumor cells were positive for vimentin, alpha-smooth muscle actin and HMB-45, consistent with epithelioid angiomyolipoma. MIB-1-labeling index was 1.6%. CONCLUSION: When one sees atypical epithelioid tumor cells in a tuberous sclerosis patient during an intraoperative consultation, one must consider epithelioid angiomyolipoma.     

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.     

Brain-derived neurotrophic factor shows a protective effect and improves recovery of the ERG b-wave response in light-damage

We investigated the neuroprotective effects of brain-derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light-induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a-wave amplitude, but there was a statistically significant difference in the b-wave amplitudes between BDNF-treated and control eyes from day 0-14 of the recovery period following 2 days of light exposure (p < 0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.