Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

C NMR study on peracetylated derivatives of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative

Peracetylated samples of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative were studied by ¹³C NMR. The acetyl carbonyl carbon signal in peracetylated β-CD was resolved into a triplet, and the three peaks were assigned by long-range C---H COSY and INAPT techniques. The individual carbonyl peak was found to be indicative of the location of the acetyl group on the 2, 3, and 6 position in the glucose residues. An acetylated derivative of a partly methylated β-CD was also subjected to ¹³C NMR analysis to determine the distribution of acetyl and, subsequently, methyl groups on the glucose residues.     

ADP-Ribosylation of Histones in Nuclei Isolated from Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

Two-dimensional electrophoresis (2D-PAGE) of a histone fraction isolated from nuclei of embryos of the sea urchin Hemicentrotus pulcherrimus exhibited almost all histone species at all stages examined. At the gastrula stage, a spot of H1A became evident and three spots closely associated with one another were found in place of a single spot of H2A.1. In the histone fraction isolated from [adenylate-³²P] NAD⁺-treated nuclei of all stages examined, autoradiograms of 2D-PAGE exhibited spots of mono [ADP-ribosyl] ated H1 and polymodified H2B.2, H3.1, H3.3 and H4 but did not show ADP-ribosylated H2A.1, H2A.2 or H2B.1. Poly [ADP-ribosyl] ated H3.2, found in morulae, was not detectable in blastulae and gastrulae. Treatment with dimethylsulfate, known to activate ADP-ribosylation in other cell types, induced poly [ADP-ribosyl] ation of H2A.2 and H2B.1 in embryos at all stages examined, and also polymodification of H3.2 in gastrulae. ADP-ribosylation of H1, H2B.2, H3.1 and H3.3 was hardly affected by dimethylsulfate treatment, though modification of H4 was blocked by this treatment. Probably, strong regulation of ADP-ribosyltransferase reactions causes failures of modification of H2A.2 and H2B.1 throughout early development and also of H3.2 at the gastrula stage. Regulation of histone ADP-ribosylation is thought to alter chromatin structures and the rate of gene expression, contributing to cell differentiation.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium