Identification of CgeA as a glycoprotein that anchors polysaccharides to the spore surface in Bacillus subtilis.

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers.

In situ dynamics of cyst and vegetative cell populations of the toxic dinoflagellate Alexandrium catenella in Ago Bay, central Japan.

Temporal changes in the in situ germination flux of cysts and the abundance of vegetative cells of the toxic dinoflagellate Alexandrium catenella were investigated in Ago Bay, central Japan from July 2003 to December 2004. The in situ germination flux (cells m-2 day-1) was measured using 'plankton emergence trap/chambers (PET chambers)'. Germination of the cysts in the sediments occurred continuously during the study, ranging from 52 to 1753 cells m-2 day-1, with no temporal trend. This germination pattern appeared to be promoted by a short mandatory dormancy period for newly formed cysts coupled with a broad temperature window for germination. For the vegetative populations, high abundances (>105 cells m-2) were recorded in the water column from spring to summer and from autumn to early winter. The size of the vegetative populations did not correlate with the cyst germination flux but rather larger vegetative populations were often observed when the water temperature was around 20°C, indicating that bloom development was mainly regulated by the temperature. Nonetheless, the continuous germination pattern of A. catenella is advantageous enabling the germinated cells to immediately exploit favorable bloom conditions.

Chronic vardenafil treatment improves erectile function via structural maintenance of penile corpora cavernosa in rats with acute arteriogenic erectile dysfunction.

INTRODUCTION: Chronic phosphodiesterase type 5 inhibitor treatment may be useful in reversing erectile dysfunction (ED). However, the mechanisms of this improvement remain unknown. AIM: The aim of this article was to determine the mechanisms of the improvement by chronic vardenafil treatment for acute arteriogenic ED in rats. METHODS: Eight-week-old male Wistar-ST rats were divided into four groups: sham-operated rats (Control group) and rats with acute arteriogenic ED induced by ligating bilateral internal iliac arteries (Ligation group), subsequently treated with low-dose (0.4 mg/kg/day; VL group) or high-dose (4.0 mg/kg/day; VH group) vardenafil for 20 days from 1 week after ligature. MAIN OUTCOME MEASURES: Erectile function was assessed based on changes of intracavernous pressure (ICP) followed by electrostimulation of the cavernous nerves and was evaluated by the area under the curve of ICP/area under the curve of mean arterial pressure (area of ICP/MAP). Transforming growth factor (TGF)-ß(1), vascular endothelial growth factor-A, endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS mRNA expression levels in penile corpus cavernosum were determined by real-time PCR. Western blotting for TGF-ß(1) protein levels and Masson trichrome staining of penile tissues were performed in each at group 4 weeks after surgery. RESULTS: In the VH group, area of ICP/MAP was significantly improved when compared with the Ligation group (P < 0.01). The smooth muscle (SM)/collagen ratio in the VH group was significantly higher than in the Ligation group (P < 0.05), and was comparable with that in the Control group. TGF-ß(1) mRNA and protein levels in the VH group were significantly lower when compared with the Ligation group (P < 0.05). CONCLUSIONS: Chronic vardenafil administration ameliorates impairment of penile hemodynamics and maintains normal SM to collagen ratio in cavernous tissues after acute arterial injury in rats.

Diastereomer-specific effects of double-stranded peptides conjugated with -L-Tyr-L-Phe- or -L-Tyr-D-Phe- residues on tyrosine phosphorylation and inhibition of src(ts)NRK, A431, MCF-7, and DU145 cell growth.

The interaction between growth inhibition and chirality, especially of diastereomers, has an important modifying effect on cancer cell proliferation. Previously, we have reported on the design, synthesis, and chemical properties of a series of novel, double-stranded peptides, (y-AA-x-AA)(2)-(CH(2))(12), with -y-AA-x-AA- and -z-AA-y-AA-x-AA- sequences conjugated to the spacer. Here, we extend those results by showing that (D-, L-) and (L-, D-) diastereomers are more potent inhibitors of tyrosine phosphorylation than (L-, L-). Although the replacement of the L-Phe-L-Phe sequence with L-Tyr-L-Phe produces a less active inhibitor, the double-stranded peptide conjugated with L-Tyr-D-Phe is more active than that conjugated with L-Tyr-L-Phe. In addition, we show that SDS-PAGE gel profiles of tyrosine phosphorylation following treatment with bis(y-Tyr-x-Phe)-N,N-dodecane-1,12-diamine appear very similar to profiles of tyrosine phosphorylation following treatment with an analog of the tyrosine kinase inhibitor, erbstatin. Moreover, the level of autophosphorylation of the epidermal growth factor receptor kinase domain (EGFRKD) treated with bis(L-Tyr-D-Phe)-N,N-dodecane-1,12-diamine was lower than that seen following treatment with bis(L-Phe-D-Phe)-N,N-dodecane-1,12-diamine. These data provide new insights for the control of cancer cell proliferation through drug designs which replace the less active -L-Phe-L-Phe- (and -D-Phe-L-Phe-) with the more active -L-Tyr-L-Phe- (and -L-Tyr-D-Phe-) sequence.

Diastereomeric selective effects of double-stranded peptides conjugated with -Phe-Phe- residue for growth inhibition and permeability of Ca(2+) on A431, src(ts)NRK, and A549 cells proliferation.

Our aim in this study is to elucidate the correlations between inhibition and chirality, especially, diastereomer, against cell proliferation of double-stranded peptides. In previous studies, we reported on the design, synthesis, and chemical properties on a series of novel double-stranded peptides, (y-AA-x-AA)(2)-spacer(S) (AA=amino acid, S=spacer, symbols x and y represent L- or D-forms, and (y-, x-) as represent of the symbol) conjugated with -y-AA-x-AA- and -z-AA-y-AA-x-AA- sequences to a spacer of carbon number n. The inhibition of A431 and src(ts)NRK cells growth by four diastereomers of the N(1),N(12)-bis(y-Phe-x-Phe)dodecanediamines (n=12) increased in the following order: (L-, L-)<(D-, D-)<(L-, D-)<(D-, L-). A similar trend was seen in the order for the activity of (y-AA-x-AA)(2)-spacer(S) with a spacer of carbon number n=2, 3, 4, 5, 6, and 12 against the cell growth inhibition. To understand the mechanism of diasteromer selective cell growth inhibition, the correlations between chirality and cell growth inhibition were investigated from the measurement of the changes in cytosolic Ca(2+) concentration (=[Ca(2+)](c)) of A431 cells. Although less active N(1),N(12)-bis(L-Phe-L-Phe)dodecanediamine increases cytosolic [Ca(2+)](c), more active diasteromers, N(1),N(12)-bis(L-Phe-D-Phe)dodecanediamine and N(1),N(12)-bis(D-Phe-L-Phe)dodecanediamine, decrease cytosolic [Ca(2+)](c) in A431 cell. This study provides diastereomeric effected new insights - this controls the polarity of double-stranded peptides - into the control of tumor cell proliferation and design of the uptake by penetration through the cell membrane of drugs.