Ethanol- and nicotine-induced membrane changes in embryonic and neonatal chick brains.

In order to study the effects of EtOH and/or nicotine on brain membrane fatty acid composition, various concentrations of EtOH and/or nicotine were injected into the air sac of chicken eggs at 0 days of incubation. Controls were injected with saline. Experimental groups were injected with either 200 micromol EtOH/kg egg, 100 micromol nicotine/kg egg, 200 micromol nicotine/kg egg, 200 micromol EtOH/kg and 100 micromol nicotine/kg egg, or 200 micromol EtOH/kg and 200 micromol nicotine/kg egg. In all experimental groups, EtOH- and nicotine-induced decreases in brain long-chain polyunsaturated membrane fatty acids were observed in stage 44 embryos, stage 45 embryos, and neonatal chicks. These EtOH- and nicotine-induced decreases in brain membrane polyunsaturated fatty acids correlated with elevated levels of brain lipid hydroperoxides and reduced brain acetylcholinesterase (AChE; EC. 3.1.1.7) activities.

P53 protein in 204 patients with primary breast carcinoma--immunohistochemical detection and clinical value as a prognostic factor.

In a retrospective study, 204 formalin-fixed and paraffin-embedded biopsies of primary breast carcinomas were tested immunohistochemically for the expression of p53 protein (PAb 1801). 38% of the carcinomas were positive with respect to p53. The expression of p53 correlated significantly with the loss of tumor differentiation (P = 0.013), but not with menopausal status, patients' age, tumor size, axillary lymph node involvement or hormone receptor status. The influence of p53 expression on prognosis was evaluated in 197 patients (T1-4 N0-2 M0, median observation time 72 months). Detection of p53 protein was associated with a significantly longer disease-free survival in node-positive women (P = 0.03). However, p53 protein did not prove to be a prognostic factor in node-negative patients. The results demonstrate the prognostic value of p53 expression in breast cancer which appears to be limited to patients with node-positive tumors.

Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA.

The Rev trans-activator of human immunodeficiency virus type 1 (HIV-1) is a protein that regulates the simultaneous appearance in the cytoplasm of both spliced and unspliced forms of viral mRNAs from the same viral transcripts by way of recognition of a target sequence termed the Rev-responsive element (RRE). Whether Rev acts directly on RNA export or by inhibition of splicing, or both, is still a matter of debate. We have addressed this issue in Xenopus laevis oocytes by microinjecting RNA molecules containing RRE along with purified recombinant Rev protein into the oocyte nuclei. Adenovirus pre-mRNA containing an RRE in the intron was spliced equally well in the absence and presence of Rev protein. Only in the presence of Rev was non-spliced pre-mRNA exported from the nucleus; more surprisingly, the excised intron lariat (containing RRE) was also exported. Furthermore, an RRE-containing mRNA molecule that lacked intron sequences was also efficiently exported from the nucleus in a Rev-dependent manner. Therefore our results demonstrate that Rev can act directly at the level of nuclear export, independent of any inhibitory effect that it may exert on the splicing of pre-mRNA. Finally, our finding that the Rev mutant M10, shown previously to be inactive in human lymphoid cells, was also unable to export RRE-containing RNA molecules from oocyte nuclei suggests that one or more cellular factors, evolutionarily conserved between humans and Xenopus, interact with Rev in both cell systems to promote nuclear RNA export.

Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides.

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

Lithium counteracts histamine suppression of human T cell mitogenesis.

Human T cell proliferative responses to concanavalin A (conA) were suppressed by approximately 50% by histamine (100 microM). In contrast, LiCl (1 or 3 mM) potentiated T cell responses by about 50%, but 10 mM LiCl had no significant effect on T cell proliferation. Histamine suppression was not significantly affected by the presence of potentiating concentrations of LiCl, whereas 10 mM LiCl completely abrogated histamine suppression.

Enzyme immunoassay (ELISA) for detection of Clostridium difficile toxin B in specimens of faeces.

Antisera against Clostridium difficile toxin B were prepared in sheep and rabbit and were used in indirect and sandwich enzyme-linked immunosorbent assays (ELISA) for the detection of toxin B. Polyvinyl chloride and polystyrene microtitration plates were tested as solid phases for the assay. Both assays had a lower limit of detection for toxin B of 1 ng/ml. They were used to detect the presence of toxin B in 210 human faecal specimens and also in the culture supernatant fluids of C. difficile strains isolated from the faecal samples. There was a close correlation between the results of sandwich ELISA and those of cytotoxicity tests and isolation of C. difficile. Our sandwich ELISA method seems to be useful as a presumptive test for detection of C. difficile toxin B.

Lack of effect of verapamil and MDL 646, a cytoprotective PGE1 analogue on cyclosporin A nephrotoxicity in vitro.

Nephrotoxicity is the most troublesome side effect of cyclosporin A (CSA) therapy. In vitro studies have shown that verapamil and PGE analogues can protect human and rat kidney allografts and murine pancreatic B cells from CSA cytotoxicity. The LLC-PK.1 pig kidney cell line has recently been shown to be a use-ful in vitro model system for studying CSA nephrotoxicity. We have adopted the MTT assay to assess the effects of CSA on LCC-PK.1 cell growth and have examined the effects of verapamil and MDL 646, a cyto-protective PGE1 analogue, on CSA nephrotoxicity. The results show that neither compound prevents the cytotoxic effects of CSA on kidney cells.