Hyaluronidase expression in cultured growth plate chondrocytes during differentiation

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.This research was supported by Grants-in-Aid (no. 11557166) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan     

Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell line

Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.     

A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.     

Inbreeding depression of female fecundity by genetic factors retained in natural populations of a male-haploid social mite (Acari: Tetranychidae)

We previously determined that certain recessive genes decrease female fecundity in a haplo-diploid spider mite, Stigmaeopsis miscanthi (Saito). However, whether the depression was caused by the breakdown of heterosis or the expression of deleterious genes retained in a population could not be determined, because we had started our inbreeding experiment from a mixture of two isolated populations. In order to answer this basic question, inbreeding effects on survival and fecundity were measured for eight small populations occurring far from the two initial populations. There was little depression of immature survival of inbred lineages in all populations. On the other hand, in two inbred lineages, both originating from the smallest populations, female oviposition decreased significantly with the increase of Wrights f-value, showing that mildly deleterious genes are actually retained even in natural populations of haplo-diploid organisms.     

Where does male-to-male “aggression” compromise “cooperation”?

We discuss how the diverse nature of aggression and cooperation can be understood if we focus our attention on where aggression reaches a compromise with non-aggression and/or cooperation in response to the relatedness between interactors. First we address whether Hamiltons rule explains the variation in male-to-male aggressiveness. Next we show that the variation in aggression and cooperation known in males of social spider mites (Saito, Evolution 49:413–417, 1995) can be explained by the change in relatedness (i.e. inclusive fitness) and effect of cooperative defence (synergistic effect). Then we learn that there is a sufficient condition of cooperation, which is determined primarily by two factors: the relatedness and synergistic effect of males. Furthermore, we expect that there is a condition where the aggression between males varies, depending upon how close the values of relatedness are to those of the sufficient condition of cooperation.This article is a continuation of the special feature Conflict among related individuals published in vol 46, pp 221–241, edited by E. Kasuya.     

DkkL1 (Soggy), a Dickkopf family member, localizes to the acrosome during mammalian spermatogenesis

Dickkopf-like 1 (DkkL1) is related to the Dickkopf gene family, a group of proteins that are characterized as secreted antagonists of Wingless (Wnt) signal transduction proteins. DkkL1 mRNA is found in preimplantation mouse embryos and in developing neural tissue, but in adults it is found primarily in the testes. In an effort to elucidate its function, the distribution of DkkL1 protein in mouse testis and mature sperm was analyzed by immuno-histochemistry and immuno-blotting techniques. DkkL1 first appeared in the developing spermatocytes in seminiferous tubules as early as Stage XII, coincident with the appearance of DkkL1 mRNA. Surprisingly, however, DkkL1 localized to the developing acrosome in spermatocytes and spermatids and to the acrosome in mature sperm. Furthermore, DkkL1 was N-glycosylated in the testis, but it did not appear to be excreted, and the DkkL1 in mature sperm was no longer N-glycosylated, suggesting that additional post-translational modifications occurred during the final stages of spermatogenesis. These results identify a member of the Dickkopf family as a novel acrosomal protein that may be involved in acrosome assembly or function, a unique role for a secreted signaling molecule.     

In vitro selection of RNA aptamers against the HCV NS3 helicase domain

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct domains, protease and helicase, that are essential for HCV proliferation. Therefore, NS3 is considered a target for anti-HCV treatment. To study RNA aptamers of the NS3 helicase domain, we carried out in vitro selection against the HCV NS3 helicase domain. RNA aptamers obtained after eight generations possessed 5' extended single-stranded regions and the conserved sequence (5'-GGA(U/C)GGAGCC-3') at stem-loop regions. Aptamer 5 showed strong inhibition of helicase activity in vitro. Deletion and mutagenesis analysis clarified that the conserved stem-loop is important and that the whole structure is needed for helicase inhibition. We compared the inhibition of helicase activity between aptamer 5 and 3'+-UTR of HCV.