Suppression of cellular invasion by glybenclamide through inhibited secretion of platelet-derived growth factor in ovarian clear cell carcinoma ES-2 cells

It has been demonstrated that potassium channels (K(+) channels) play significant roles in some malignant phenotypes. Here, we provide the first evidence that treatment with glybenclamide, an ATP-sensitive K(+) channel blocker, inhibited cell migration in an ovarian clear cell carcinoma cell line, ES-2. Treatment with glybenclamide or knockdown by siRNA targeted against K(+) channel subunits demonstrated the suppression of ovarian cancer cell invasion, which occurred via inhibition of PDGF-AA secretion. Therefore, our findings suggest that K(+) channel blockers may be useful chemotherapeutic drugs for blocking the invasiveness of ovarian cancers.     

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.     

Inhibitors of human 2,3-oxidosqualene cyclase (OSC) discovered by virtual screening

Five human 2,3-oxidosqualnene cyclase (OSC) inhibitors were discovered using the combination of a virtual screening based on a docking study and an isotope-free assay system. All of these inhibitors were revealed to have activities comparable or superior to that of LDAO, a known OSC inhibitor. The computational study of the newly identified inhibitors suggested that CH/π interactions with F444 and W581 contribute to the binding, and these interactions are candidates for additional structural filters in the next generation of virtual screening.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis

We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.     

Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1

CCN3/NOV activates the Notch signal through the carboxyl terminal cysteine-rich (CT) domain. CCN3 transfection to Kusa-A1 inhibited osteogenic differentiation and cell proliferation, which is accompanied by upregulation of Hes/Hey, Notch downstream targets, and p21, a CDK inhibitor. Upregulation of Hes/Hey and p21 was abrogated by the deletion of CT domain. Anti-proliferative activity of CCN3 was also abrogated by CT domain deletion whereas anti-osteogenic activity was not completely abrogated. We found that CT domain-deleted CCN3 still possesses antagonistic effect on BMP-2. These results suggest that CCN3 employs Notch and BMP pathways in anti-osteogenic activity while it inhibits cell proliferation uniquely by Notch/p21 pathway.     

Dioxin interferes in chromosomal positioning through the aryl hydrocarbon receptor

Each chromosome occupies its own-specific space called a ‘territory’ within the interphase nucleus, and the arrangement of chromosome territories (CTs) is important in epigenetic mechanisms. The molecular mechanism to determine the positioning of CTs, however, remains unknown. On the other hand, dioxin is known to be the typical environmental pollutant that affects a wide variety of biological events in many species. Here, we show that dioxin enlarges the minimum distance between chromosome 12 and chromosome 16 territories in human preadipocyte cells, and the alteration of chromosome positioning is canceled by an aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. Thus, AhR may be a key molecule to regulate chromosome positioning. Our results suggest a novel effect of dioxin toxicity, and demonstrate a clue to reveal the novel molecular mechanism for the arrangement of CTs.