Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion.

We have used the human Lewis blood group fucosyltransferase cDNA and cross-hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1----4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x-negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM-1-dependent cell adhesion.     

Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.     

Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis.

A 3.8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance. The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light. E. coli containing the 3.8 kb PstI fragment produced a 38-40 kDa protein which cross-reacted with antibodies raised against the E. coli RecA protein. The cloned DNA thus probably encodes a RecA homologue.     

Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves

Glutamate semialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea (Pisum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 mumol.min-1.mg-1; 23 nkatal.mg-1) is very much higher than previously reported.     

Synergistic effect of heat-shock and UV-irradiation on mitosis and protein synthesis in G2-phase plasmodia of Physarum polycephalum Schw--reduction in UV-induced mitotic delay in a preheat-shocked system

The synergistic effect of UV irradiation and heat-shock during the last 3 hr of G2 phase of the cell cycle in the plasmodia of P. polycephalum, in terms of mitotic delay and inhibition of protein synthesis, has been evaluated. The mitotic delay due to both perturbers coordinately increased closer to mitosis. Maximum mitotic delay was obtained in plasmodia heat-shocked after UV irradiation, indicating the presence in this system of either a heat-labile mitogenic substance which is comparatively less susceptible to UV or a substance which is made more susceptible to hyperthermia by UV. A protective role for heat-shock applied before irradiation has been observed in that, radiation-induced mitotic delay is significantly reduced in this combination. There was severe inhibition of translation in all the perturbed classes. Organelle level effects which are independent of major protein synthetic activities or different levels of heat-shock protein production could be the reason for the lack of correlation between percentage inhibition of general protein synthesis and the extent of mitotic delay with respect to the two double-perturbed systems.     

Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein.

In this paper, we report the cloning and analysis of a cDNA encoding a protein of M(r) congruent to 47,000 (p47), which is localized to the nucleus of rat hepatocytes. The cDNA showed 37% overall sequence identity with a mouse translation initiation factor, eIF-4A, which belongs to a family of putative ATP-dependent RNA helicases. We raised polyclonal antibodies against the fusion protein and by indirect immunofluorescence on primary cultures of hepatocytes have demonstrated that p47 is located in the nucleus. Although only approximately 27% of hepatocytes showed this nuclear staining, most of the nuclei in proliferating transformed cell lines such as 3T3, PtK-1, and Hela were fluorescently labeled. Studies on serum-starved cells in culture indicated that p47 was expressed in a cell cycle-dependent manner. Northern analyses demonstrated that the levels of p47 mRNA were high in fetal liver and dropped significantly after birth to low levels in adult liver. Our data suggest that p47 is developmentally regulated in rat liver at the mRNA level.     

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.     

Assessment of the genotoxic potential of riboflavin and lumiflavin. B. Effect of light.

On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the umu test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.     

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)