Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.     

Insulin-stimulated alpha-(methyl)aminoisobutyric acid uptake in skeletal muscle. Evidence for a short-term activation of uptake independent of Na+ electrochemical gradient and protein synthesis.

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.     

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.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

Deacylated lipopolysaccharide inhibits plasminogen activator inhibitor-1, prostacyclin, and prostaglandin E2 induction by lipopolysaccharide but not by tumor necrosis factor-alpha

Bacterial LPS and TNF induce vascular endothelial cells to express a variety of response molecules. LPS that is partially deacylated (dLPS) by a human neutrophil enzyme blocks the ability of LPS, but not TNF, to augment one of these responses, the expression of endothelial cell surface molecules that promote neutrophil adherence (J. Exp. Med. 1987; 165:1393-1402). We show that dLPS can inhibit the ability of LPS, but not TNF, to elicit the expression of plasminogen activator inhibitor-1 (PAI-1), prostacyclin, and PGE2 by human umbilical vein endothelial cells. dLPS also prevented the accumulation of specific PAI-1 mRNA in response to LPS, but not to TNF. Neither the LPS- or TNF-induced expression of PAI-1 nor the dLPS inhibition of the LPS response was mediated by prostanoids. These results indicate that dLPS can specifically block a variety of endothelial cell responses to LPS and provide support for the hypotheses 1) that dLPS and LPS may interact with a common target molecule on or in endothelial cells, and 2) that dLPS, produced by enzymatic deacylation of LPS in vivo, could inhibit endothelial cell stimulation by LPS and thereby limit the host inflammatory response to invasive gram-negative bacteria.     

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.