Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.     

Plasminogen activator inhibitor-type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture.

Previous studies have shown that a glycoprotein of Mr 47,000 (designated Gp47) is a major biosynthetic product of retinal endothelial cells in vitro (Canfield, Schor, West, Schor & Grant (1987) Biochem. J. 246, 121-129). We now present data indicating that (a) an identical protein is secreted by bovine retinal pericytes, (b) this protein is plasminogen activator inhibitor-type I (PAI-1), as revealed by immunoprecipitation with specific antibodies and reverse fibrin zymography, and (c) retinal endothelial cells and pericytes synthesize different species of matrix macromolecules, that is: type IV collagen is the major collagen secreted by endothelial cells, whereas pericytes produce predominantly type I collagen; fibronectin and thrombospondin are synthesized by both cell types. Our studies also indicate that PAI-1 is produced, albeit at considerably lower levels, by large vessel vascular cells (aortic endothelial and smooth muscle cells) and human skin fibroblasts. PAI-1 produced by human skin fibroblasts appears to be a distinct molecular species compared to its bovine counterpart as assessed by its slower mobility on SDS/polyacrylamide-gel electrophoresis. The potential significance of elevated PAI-1 production by retinal endothelial cells and pericytes, as well as their distinctive patterns of matrix biosynthesis, is discussed in terms of the involvement of these cells in the maintenance and remodelling of microvessel basement membrane.     

Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes.

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.     

In vitro growth properties of Xenopus retinal neurons undergo developmental modulation

To determine whether Xenopus retinal neurons undergo intrinsic developmental changes in growth properties, retinal explants from embryos and tadpoles of different stages were grown on laminin, fibronectin, and collagen I in serum-free media. Growth was assayed in terms of a neurite growth index (NGI) and the appearance of clockwise bundles, or a clockwise growth index (CGI). The first neurites from stage 25 optic vesicles are pioneers and display a unique growth phenotype; they emerge rapidly, survive for a short time, show little substrate preferences for growth (they grow almost as well on BSA as they do on laminin and fibronectin), and form no clockwise bundles under any conditions. Neurites from progressively older retinas (stages 32-37) share with stage 25 neurites the rapid outgrowth pattern, but begin to show substrate preferences and clockwise growth. From stage 40 to 50, the mature growth pattern is expressed; a lag in initial outgrowth, long-term survival, distinct substrate preferences (they grow 10 times better on laminin and fibronectin than on BSA) and display robust clockwise growth patterns on laminin and fibronectin. The acquisition of clockwise growth is independent of optic fiber contact with the tectum or exposure to diffusible factors from mature brain tissues. The results suggest that retinal neurons undergo developmental modulation of surface adhesive properties and/or cytoskeletal organization.     

Evidence for two closely related isozymes of arylamine N-acetyltransferase in human liver

Acetyl CoA-dependent arylamine N-acetyltransferase (EC 2.3.1.5) is the target of a genetic polymorphism in the metabolism of drugs and carcinogens. N-Acetyltransferase was purified 1000-fold from cytosol of human liver and its identity was verified by amino acid sequence homology of two of its tryptic peptides with published rabbit and chicken N-acetyltransferase sequences. Enzyme activity correlated with the presence of two proteins, NAT-1 and NAT-2, with indistinguishable molecular masses (31 kDa). NAT-1 and NAT-2 could be separated by anion-exchange chromatography and were functionally distinguished by their different apparent affinities for the acceptor amine sulfamethazine (SMZ). Antibodies raised against NAT-1 were able to recognize both isozymes on Western blots.     

Elastase activities of human bladder cancer cell lines derived from high grade invasive tumours

Elastase activities in intact human bladder cancer cell lines, established from three patients, were measured using a fluorogenic substrate highly specific for elastase, under conditions of physiological pH and ionic strength. This method allowed separation of cell-associated from secreted enzyme activity. As secreted elastase accounted for only 8% of the total, we concluded that the elastases were present at the cell surface. Inhibition studies using extracts of cell-surface elastases showed them to be serine proteinases which were also inhibited by alpha 1-antitrypsin. Partially purified fractions showing the highest specific activity towards the fluorogenic substrate hydrolysed insoluble elastin thus confirming the presence of elastases. This is the first time that elastase activity has been demonstrated in human bladder cancer cells and may represent a mechanism involved in tumour invasion.     

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.