Roles of hnRNP A1, SR proteins,and p68 helicase in c-H-ras alternative splicing regulation

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.     

Platelet-Activating Factor (PAF) Modulates Peritoneal Mouse Macrophage Infection by Leishmania amazonensis

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis–macrophage infection was inhibited by PMA in all systems tested. Received: 27 September 2000 / Accepted: 15 December 2000     

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonellacells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.     

Glutathione S transferase M1 andT1 genotypes and susceptibility to smoking related larynx cancer

Susceptibility to smoking related larynx cancer has been suggested to be associated with genetically determined differences in the ability to detoxify carcinogens present in tobacco smoke. The genetic polymorphisms of glutathione S-transferases, involved in the metabolic inactivation of, for example, tobacco derived carcinogens, have been recognized as potential risk modifiers in various environmentally induced malignancies, including larynx cancer. We employed PCR-based methods to determine the distribution of the GSTM 1 and G STT1 null genotypes in 171 larynx cancer patients and 180 controls to examine further their potential role in individual susceptibility to this neoplasm. The GSTM 1 null genotype was found in 49 1 % of the cases and 57 7 % of the controls and the GSTT1 null genotype in 17 5 % of the cases and 21 7 % of the controls, respectively. Larynx cancer risk associated with the lack of GST M 1 (OR = 0 7; 95 % CI: 0 5-1 1) or GSTT1 (OR = 0 8; 95 % CI: 0 5-1 3) was not significantly affected by age, smoking status, or cancer progression. Although this study thus suggests no role for the G STM 1 and GSTT1 gene polymorphisms in individual susceptibility to smoking-related larynx cancer, due to its relatively small sample size more data are required before any definite conclusions can be drawn.     

Effect of nitric oxide synthase inhibition on hypercapnia-induced hypothermia and hyperventilation

Hypercapnia elicits hypothermia in a numberof vertebrates, but the mechanisms involved are not well understood. Inthe present study, we assessed the participation of the nitric oxide(NO) pathway in hypercapnia-induced hypothermia and hyperventilation bymeans of NO synthase inhibition by usingN-nitro-L-arginine(L-NNA). Measurements ofventilation, body temperature, and oxygen consumption were performed inawake unrestrained rats before and afterL-NNA injection(intraperitoneally) and L-NNA injection followed by hypercapnia (5%CO₂). Control animals received saline injections. L-NNA alteredthe breathing pattern during the control situation but not duringhypercapnia. A significant (P < 0.05) drop in body temperature was measured after bothL-NNA (40 mg/kg) and 5%inspired CO₂, with a drop inoxygen consumption in the first situation but not in the second.Hypercapnia had no effect onL-NNA-induced hypothermia. Theventilatory response to hypercapnia was not changed byL-NNA, even thoughL-NNA caused a drop in bodytemperature. The present data indicate that the two responses elicitedby hypercapnia, i.e., hyperventilation and hypothermia, do not share NOas a common mediator. However, theL-arginine-NO pathwayparticipates, although in an unrelated way, in respiratory function andthermoregulation.

     

Cellulolytic enzymes for the hydrolysis of leached beet cosette

Summary Cellulolytic fungi were isolated from rotting leaves and tested for extra-cellular cellulase activities (CMCase, avicelase, cellobiase and xylanase). The effect of the proportion of the enzyme activities on the rate of degradation of leached beet cosette was observed using a range of supernatant fluids in appropriate combinations. At low cosette concentrations (1.5–3.0 g/l), avicelase and cellobiase were the rate limiting enzymes; avicelase in the initial stages of reaction and cellobiase after 6–8 hours, when cellobiose inhibition becomes important. A ratio of celiobiase to avicelase of approx 2.0 was established as appropriate. At higher substrate concentrations (10 g/l, 40 g/l) the best cellobiase to avicelase ratio was maintained and up to 40% hydrolysis was obtained in the 10 g/l incubation with 10 Uav/l and 20 Ucellob/l. At 100 g/l cosette concentration, substrate inhibition was observed.     

Bioenergetics of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Cells Grown at Alkaline Conditions

Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4–3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.