The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells

CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, and adhesion. In addition, it has been known that CD9 can associate with other proteins. Here we demonstrated the physical and functional association of CD9 with epidermal growth factor receptor (EGFR) on MKN-28 cells. Double-immunofluorescent staining and immunoprecipitation demonstrated the complex formation of CD9-EGFR and CD9-beta(1) integrin, and that both complexes are colocalized on the cell surface especially at the cell-cell contact site. Anti-CD9 monoclonal antibody ALB6 induced a dotted or patch-like aggregation pattern of both CD9-EGFR and CD9-beta(1) integrin. The internalization of EGFR after EGF-stimulation was significantly enhanced by the treatment with ALB6. CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO-HER/CD9), which were transfected with pTJ/human EGFR/CD9. Furthermore expression of CD9 specifically attenuated EGFR signaling in CHO-HER/CD9 cells through the down regulation of surface expression of EGFR. These results suggest that CD9 might have an important role that attenuates EGFR signaling. Therefore, CD9 not only associates EGFR but also a new regulator, which may affect EGF-induced signaling in cancer cells.     

Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.     

Effect of Single Nucleotide Polymorphisms of Toll-Like Receptor 4 (TLR 4) on Reproductive Performance and Immune Function in Dairy Cows

In dairy cows, inflammatory diseases caused by infection with pathogenic bacteria post calving affect ovarian functions. This study examined the relationship between single-nucleotide polymorphisms (SNPs) of Toll-like receptor 4 (TLR4), reproductive performances [the number of artificial insemination (AI) application and days open], and immune cell functions (apoptosis and migration). Two hundred Holstein cows from the Obihiro University farm were included. The SNPs of TLR4 were genotyped by PCR-restriction fragment length polymorphism (PCR–RFLP) method. Polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. The number of AI application in the animals with T/C genotype in the TLR4 exon3 was lower than that in animals with C/C genotype (1.6 ± 0.2 and 2.2 ± 0.2, respectively). Among the animals with TLR4 exon3 polymorphisms, the days open was shorter for the T/C cows than that for C/C cows (100.7 ± 6.9 days and 136.6 ± 9.0 days, respectively). The SNPs in the TLR4 intron did not affect the number of AI and days open. The apoptosis percentage of PMNs treated with lipopolysaccharide (LPS; 0.001 and 1 μg/ml) tended to be lower in the T/C genotype compared to that in the C/C genotype. The transmigration rates of PMNs, and IL-1β production in PBMCs were tended to be higher for the animals with the T/C genotype compared to those for animals with the C/C genotype. Taken together, these results suggest that TLR4 polymorphisms offer a meaningful tool to judge the reproductive potential and immune activity in individual cows.     

Effect of sucrose on ascorbate level and expression of genes involved in the ascorbate biosynthesis and recycling pathway in harvested broccoli florets

The relationship between sucrose (Suc) and ascorbate (AA) metabolism was investigated in harvested broccoli (Brassica oleracea L. var. italica) florets. Decreases in both Suc and AA content were observed in broccoli florets 48 h after all the leaves were excised, but none were observed when the plants were kept intact or with leaves attached in a room at 20 degrees C. In harvested broccoli plants without leaves and roots, continuous absorption of a 10% (w/v) Suc solution from the cut surface of the stem suppressed the degreening of sepals and the loss of AA content in florets. The expression of the genes related to AA metabolism in chloroplasts and its biosynthesis were up-regulated by Suc feeding in broccoli florets. These data suggest that a decline in Suc leads to considerable damage not only to AA biosynthesis but also to the hydrogen peroxide-scavenging system in chloroplasts. In addition, the cessation of the Suc supply from leaves can be the main factor of AA degradation in harvested broccoli florets.     

TIME-LAPSE ANALYSES OF SEXUAL ISOLATION BETWEEN TWO CLOSELY RELATED MATING GROUPS OF THE CLOSTERIUM EHRENBERG SPECIES COMPLEX (CHLOROPHYTA)1

Sexual isolation between Groups A and B of Closterium ehrenbergii, two closely related species, was studied by a multiple-choice mating method, as well as the nochoice mating method which has been used in previous work on microalgae. Time lapse photomicrographs and the difference in cell shape and size between the two mating groups allowed identification of a given cell in the mixture as either Group A or B, even when certain morphological changes occurred during the several day culture required for sexual induction. When plus and minus mating types of Group A were mixed with those of Group B (multiplechoice mating), no intergroup hybrid zygospores were formed. However, many intragroup zygospores of either Group A or B were formed. When one plus strain of Group A was mixed with one minus strain of Group B or when one plus strain of Group B was mixed with one minus strain of Group A (no-choice mating), intergroup sexual interactions took place resulting in a small number of hybrid zygospores; however, the process took much longer than intragroup sexual interactions. It was also shown that cell size difference itself hardly affects sexual interactions between haploid and autodiploid strains of Group A. It is suggested that sexual isolation between Groups A and B would be complete in nature, although they may interact sexually in the laboratory.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

The fragility of a structurally diverse duplication block triggers recurrent genomic amplification

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12–21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.     

Morphological and cytogenetic characteristics of intergroup hybrids between closely related mating groups of the Closterium ehrenbergii species complex (Desmidiales, Chlorophyta)

Five F₁ hybrid strains were established from rare survivors in intergroup crosses between three closely related mating groups (A, B and H) of the Closterium ehrenbergii Meneghini ex Ralfs species complex. Cell sizes of these five strains studied under our standard culture conditions were compared to those of their parental stains and also to the total range of cell-size variation in each mating group. All five F₁ strains were larger in mean cell width than their parental strains. In cell length, three of them were larger than, one was the same as, and the other was intermediate between their parental strains. Their cell sizes were always larger than the range of their respective smaller parental mating group and three of them were larger than the range of their respective larger parental mating group.