Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.     

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.     

Computer-assisted imaging cytometry of nuclear chromatin reveals bone tumor virus infection and neoplastic transformation of adherent osteoblast-like cells

Established osteoblast-like (OB) cells infected with the bone tumor-inducing C-type retrovirus OA MuLV remained nontumorigenic over 104 cell culture passages. DNA histograms revealed a new cell population with a stem line peak at 5c. A second OA MuLV-infected OB cell line underwent neoplastic transformation with increasing passage level. These cells showed diffuse aneuploidy. Stepwise linear discriminant analysis of the chromatin structure of control, OA MuLV-infected, and FBR osteosarcoma virus-transformed cell lines resulted in various levels of discrimination ranging between 79.6% for control cells versus nontumorigenic OA MuLV-infected cells, and 96.6% for nontumorigenic OA MuLV-infected cells versus FBR osteosarcoma virus-transformed cells. OA MuLV-infected tumorigenic cells and FBR osteosarcoma virus-transformed cells were discriminated at a 93.6% level.     

Localization of c-ERB A proteins in rat liver using monoclonal antibodies

Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction. These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses. In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts. Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized.     

Noncovalent modulation by ATP of the acyl transfer from acyl-glyceraldehyde-3-phosphate dehydrogenase to phosphate

The effect of ATP on the formation, spectral properties, and reactions of [beta-(2-furyl)acryloyl]glyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) has been investigated. The chromophoric FA-GPDH has the advantage of providing spectrophotometric signals of the interaction of acyl enzyme with nucleotides and dinucleotides. The results are consistent with the exclusive existence of two acyl-enzyme conformations previously inferred from the interaction of the acyl enzyme with NAD+ and NADH. ATP interaction stabilizes a conformation different from that stabilized by NAD+. The inhibitory effects of ATP on these reactions are consistent with the reported inhibitory effect of ATP on the steady-state reaction with the true substrate. The physiological significance of these results to the regulation of glycolysis, via the ligand-dependent fate of 3-phosphoglycerol-GPDH, is discussed.     

Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.     

On the clonal origin of tumours--lessons from studies of intestinal epithelium

Clonal studies of adult chimaeric mouse epithelium have demonstrated the monoclonal composition of crypts of Lieberkühn. In neonatal life, however, polyclonal crypts have been found, indicating that crypts are of polyclonal origin. We here relate these findings to studies of mosaic tissues which have addressed the question whether solid tumours are of monoclonal or polyclonal origin. The issues has so far remained unresolved because the expected frequencies of polyclonal tumours, given polyclonal origins, have not previously been estimated. A general approach for the calculation of such expected values is suggested. The consistent reports of tumours with polyclonal components suggest that autocrine or paracrine mechanisms play an important role during tumorigenesis.     

New motile anaerobic bacteria growing by succinate decarboxylation to propionate

Three strains of new anaerobic, gram-negative bacteria which grew with succinate as sole source of carbon and energy were isolated from anoxic marine and freshwater mud samples. Cells of the three strains were small, non-spore-forming, motile rods or spirilla. The guanine-plus-cytosine content of the DNA of strain US2 was 52.6±1.0 mol%, of strain Ft2 63.5±1.4 mol%, and of strain Ft1 62.6±1.0 mol%. Succinate was fermented stoichiometrically to propionate and carbon dioxide. The growth yields were 1.2–2.6 g dry cell mass per mol succinate degraded. Strains US2 and Ft2 required 0.05% w/v yeast extract in addition to succinate for reproducible growth. Optimal growth occurred at 30°–37°C and pH 6.8–8.0. Addition of acetate as cosubstrate did not stimulate growth with any strain. Strain Ft2 grew only under strictly anaerobic conditions, whereas strains US2 and Ft1 tolerated oxygen up to 20% in the headspace. Strains US2 and Ft2 grew only with succinate. Strain Ft1 also converted fumarate, aspartate, and sugars to propionate and acetate. This strain also oxidized propionate with nitrate to acetate. Very low amounts of a c-type cytochrome were detected in propionate plus nitrate- or glucose-grown cells of this strain (0.4 g x g protein^-1). Moderate activities of avidin-sensitive methylmalonyl-CoA decarboxylase were found in cell-free extracts of all strains.