Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

Variability in messenger RNA levels in human umbilical vein endothelial cells of different lineage and time in culture

Summary Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plaminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

Induction of c-fos mRNA Expression by Afterdischarge in the Hippocampus of Naive and Kindled Rats

Periodic induction of focal electrical seizure [afterdischarge (AD)] is an absolute prerequisite for the development of kindling, an animal model of complex partial epilepsy. Once established, it is a permanent condition. The mechanism(s) that translate ADs, which last tens of seconds, into life-long alterations in the CNS is unclear. Cellular immediate-early genes have been implicated in the conversion of short-term stimuli to long-term alterations in cellular phenotypes by regulating target gene expression. We have investigated the contribution of one such early gene, c-fos, to this process. The relationship between ADs and expression of c-fos gene in the rat hippocampus, a key structure in kindling development, was studied by analysis of mRNA levels. The low constitutive expression of c-fos mRNA in the hippocampus was not altered by kindling. There was an "all-or-none" relationship between induction of c-fos and the duration of AD. The threshold for induction was approximately 30 s of AD. Above-threshold ADs induced c-fos in both naive and kindled animals to the same extent and with identical temporal profiles. Although the expression of c-fos is unchanged with kindling, c-fos may nonetheless contribute to many long-term changes of kindling, both adaptive and epileptogenic.     

Transformation and expression of a cloned δ-endotoxin gene in bacillus thuringiensis

Abstract A shuttle vector containing the replication region of a resident plasmid of B. thuringiensis , was used to determine the conditions allowing efficient transformation of B. thuringiensis by electroporation. Using this plasmid a δ-endotoxin gene was cloned and expressed both in Escherichia coli and B. thuringiensis . It was shown that this gene was poorly expressed in the wild type situation whereas after cloning in acrystalliferous strains of B. thuringiensis large amounts of crystal protein were obtained.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.