Localization of fetal aldolases during early stages of azo-dye hepatocarcinogenesis in rat

We have looked for the synthesis of fetal aldolases A and C during the early stages of hepatocarcinogenesis induced by 3′-methyl-4-(dimethylamino) azobenzene in the rat. Using indirect immunoperoxidase and immunofluorescence techniques we show that oval and transitional cells are the main cellular sites of fetal aldolases A and C production while hepatocytes only synthesize aldolase B. The synthesis of aldolases A and C was confirmed by electrophoresis analysis. These results indicate that different cell types are involved in fetal aldolase production during the early stages of azo-dye feeding and during regeneration after carbon tetrachloride intoxication where the synthesis of these isozymes is restricted to sinusoïdal cells.     

Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.     

Macrofaunal diversity and habitat structure in intertidal boulder fields

Low-midshore boulder fields in Europe are increasingly subject to degradation related to beach fishing for crabs and abalones. The aim of the study was to better understand the structure and species diversity of macrofaunal assemblages living in a low-midshore boulder field in order to define conservation strategies for this biotope. Sampling strategy involved different spatial scales (macro- and microstrata) relative to the complexity and heterogeneity of the habitat. Assemblages of species and the corresponding habitats were identified by multifactorial analysis and compared by ANOVAs. The results show a hierarchical organization of the macrofauna within the boulder field, corresponding to three spatial levels of habitat structure: (i) eight microhabitats at the lowest level of spatial organization, each defined by a specific assemblage (subcommunity); (ii) three habitats at a middle level combining these microhabitats, each associated with a specific community (open rock, protected rock and sediment); and (iii) three macrohabitats at the uppermost level (corresponding to the scale of the entire boulder field and including the main geomorphological features of the beach), each defined by a specific assemblage of species (boulders on boulders, boulders on bedrock, and boulders on sediment). Two microhabitats with particularly high species diversity were regarded as the most important ecological units of the field in terms of conservation of specific and functional biodiversity. Comparison of habitat/community parameters showed (i) that habitat heterogeneity was not an accurate indicator of faunal diversity, and (ii) that habitat complexity enhanced the species richness of the fauna, but only above a threshold value. This enhancement was due to semi-sheltered microhabitats, which were found only in the most complex areas of the boulder field. It is likely that this complexity affects species richness qualitatively more than by the diversity of microhabitats. In other words, a cross-scale effect is apparent in which high habitat complexity on the middle spatial scale creates microhabitats on the lowest spatial scale that are characterized by stable semi-sheltered environmental conditions conducive to a maximum of species.     

NOV/CCN3 induces adhesion of muscle skeletal cells and cooperates with FGF2 and IGF-1 to promote proliferation and survival

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin beta1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.     

A technique for labelling the sample surface for quick-freeze,deep-etch,rotary replication electron microscopy: application to the study of geletin gel structure

A method using magnesium oxide crystals to label the surface of physical gels, such as gelatin gel before quick-freezing is described and discussed. The quick-freeze, deep-etch, rotary replication technique is most adapted to 3-D visualization of physical gel structure. However, it is known that the depth which ultrarapid freezing may reach is limited by the growth of ice crystals as the distance from the surface of the specimen (rapidly cooled by smashing against a cooled metal plate) increases. Consequently, intact preservation of structures occurs only in superficial zones of the specimen. The MgO surface labelling technique provides a simple means for surface recognition. It enables the estimation of a given replicated area depth, taking into account the angle of specimen scraping before etching and replicating. By comparison of views of the same replica at different depths, freezing artifacts may be recognized even when they cause only slight deformations in the structure. This is particularly necessary for interpretation of gel network geometry: interpretation can be made with certainty only if a reliable surface reference marker exists. For gelatin gels, the depth of best freezing can be estimated to be around 5 μm from the frozen sample surface.     

Human interferon gamma receptor 1 (IFNGR1) gene maps to chromosome region 6q23–6q24

Summary The human interferon receptor (IFNGR1) gene has been localized by in situ hybridization to chromosome 6 at q23–q24. This chromosomal region is often deleted in lymphoid cell malignancies.     

Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: Evidence for little recombination in the 3'' end of the human gene

We report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. We have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions--or both of them combined--could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine4311 plus leucine2712 representing the Ag(x) epitope, and that encoding asparagine4311 plus proline2712 the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu2712/Ser4311 allele is associated both with reduced serum levels of LDL-cholesterol and apo B and with raised levels of HDL. However, these differences are of smaller effect than those associated with the XbaI RFLP of the apo B gene in this sample. We have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2712 and 4311 in all of these samples, although there are large allele frequency differences between these populations. In addition, there is strong linkage disequilibrium with allelic association between the alleles of these sites and those of the XbaI RFLP in all the populations examined. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.