Detection of multiple conformations of the E-domain of 5S rRNA from Escherichia coli in solution and in crystals by NMR spectroscopy

NMR spectroscopy of the E-domain fragment of Escherichia coli 5S rRNA indicates that this molecule exists in solution as either a stem-loop or as a duplex with two U-U base pairs in the bulge region. At temperatures below 27 degrees C, interconversion between the monomeric and dimeric forms in solution occurs on a time scale of weeks and allows the preparation of samples on which NMR structure determinations can be carried out on predominantly monomeric or dimeric species. The NMR results obtained provide comparison data for the distinction between A- and B-form E.coli 5S rRNA and for the possible kinetics of conversion between these forms. NMR evidence is presented that the duplex form also exists in crystals and suggestions are made for means to obtain stem-loop conformations of E-domain and other small RNA stem-loop sequences in crystals.     

The behavior of the coiled body in cells infected with adenovirus in vitro

The coiled body is a phylogenetically conserved nuclear organelle whose function is not known. Probes for detection of p80-coilin, an 80 kDa protein enriched in the coiled body, have made possible studies determining the behavior of the coiled body during the cell cycle, in proliferating cells, as well as reports suggesting some relationship of the coiled body to mRNA splicing and to the nucleolus. The objective of this study is to examine the distribution of p80-coilin and nucleolar proteins in cells infected with adenovirus in vitro. HeLa cells grown as monolayers were infected with successive dilutions of type 5 human adenovirus culture and fixed in methanol/acetone at different time points. Single and double indirect immunofluorescence was performed with human autoantibodies to p80-coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM-Scl, and To, as well as rabbit polyclonal serum to p80-coilin (R288) and mouse monoclonal antibody to adenovirus 72-kDa DNA-binding protein. Indirect immunofluorescence (IIF) with anti-p80-coilin antibodies showed that the usual bright dot-like coiled body staining pattern was replaced in infected cells by 1-5 clusters of tiny dots at the periphery of the nucleus. This phenomenon was first detected within 12 h of infection and affected more severely cells with increased length and load of infection. Cells subjected to heat shock presented no such alteration. Double IIF showed cells with abnormal coiled body appearance expressed the viral 72-kDa DNA-binding protein. Nucleolar proteins RNA polymerase I and NOR-90/hUBF became associated with the p80-coilin-enriched clusters and were no longer detected in the nucleolus. Other nucleolar proteins, like PM-Scl and To, remained associated to the nucleolus and were not detected in the newly formed clusters. Fibrillarin had a heterogeneous behavior, being restricted to the nucleolus in some infected cells while in some others it was associated with the p80-coilin-enriched clusters. Thus our results showed that in vitro adenovirus infection induced radical redistribution of nucleolar and coiled body constituents into newly formed structures characterized by clusters of tiny dots in the periphery of the nucleus. The fact that three major proteins involved in rRNA synthesis and processing colocalized with p80-coilin in these clusters may bring additional support to the idea that the coiled body and p80-coilin may be implicated in functions related to the nucleolus.     

The use of intermittent lighting in broiler raising. 2. Effects on the somatotrophic and thyroid axes and on plasma testosterone levels

Male and female broiler chicks were raised separately in nearly continuous lighting [23 h light (L):1 h dark (D), CL] and consumed feed ad libitum. At 7 d of age, the intermittent lighting schedule (1L:3D, IL) was imposed on half of the chicks, whereas the other chicks remained under CL. In addition to performance characteristics, several parameters of the somatotrophic and thyrotrophic axes were studied together with plasma concentrations of testosterone. Males had a higher growth rate than females regardless of the imposed lighting schedule and this pronounced growth difference is reflected by higher plasma concentrations of growth hormone (GH), and a better GH receptor occupancy. Differences in growth rate between sexes could not be attributed to differences in circulating 3,3',5-triiodothyronine (T3) levels or to hepatic deiodination activities. However, from 3 wk of age onwards, males had significantly higher plasma testosterone levels than females. Plasma GH and T3 levels decreased whereas plasma insulin-like growth factor-I and thyroxine levels increased with age in all experimental groups. The age-related decline in plasma GH levels were less pronounced for males than for females. No major changes in other hormonal parameters or deiodination activities could be observed as a result of imposing IL, except for the higher plasma GH levels of IL chickens, and for plasma testosterone concentrations in IL males at Day 41, which were twice the levels found in their CL counterparts. These results therefore suggest that the somatotrophic axis as well as circulating testosterone levels mediate the sex-related differences in growth rate and the compensatory growth as present in males.     

Thoracic lymphangiomatosis

The prognostic value of p53 protein in tumor extracts as measured by ELISA was studied retrospectively in 228 non-small cell lung cancer (NSCLC) patients. The assay measures both wild-type and mutated p53. The specimens on which this study was performed have been used earlier to analyze the prognostic impact of components of the plasminogen activation system, which enabled an analysis of relationships between these components and p53 protein. The median of the p53 protein values in the 228 patients was 0.10 (range, 0-0.70) ng/mg protein. Survival analysis comparing patients with p53 levels below versus above the median showed no significant difference (P = 0.67). When analyzing the histological types, adenocarcinoma (n = 106), squamous cell carcinoma (n = 84), and large cell carcinoma of the lung (n = 38) separately, similarly, no significant differences in survival between patients having low versus high tumor p53 levels were found. When comparing levels of p53 protein in the three histological types, a significant difference (P < 0.0001) was found, with adenocarcinomas having the lowest levels. There was a weak positive correlation (r = 0.22) between p53 protein and plasminogen activator inhibitor type 1 (PAI-1). Multivariate analysis proved no impact of p53 on survival; tumor size, PAI-1, and lymph node involvement were the only variables with significant influence on survival. These data indicate that p53 protein quantitated with a sandwich ELISA in tumor extracts from NSCLC has no prognostic value, but the observed statistically significant difference of p53 protein content between histological subgroups may be related to differences in etiology and biology in different NSCLC subtypes. In addition, the weak association found between p53 protein and the independent prognostic marker PAI-1 could suggest yet undefined interactions in lung cancer.     

Mechanisms for the transendothelial migration of HIV-1-infected monocytes into brain

HIV-1 penetration of the brain is a pivotal event in the neuropathogenesis of AIDS-associated dementia. The establishment of productive viral replication or up-regulation of adhesion molecule expression on brain microvascular endothelial cells (BMVEC) could permit entry of HIV into the central nervous system. To investigate the contribution of both, we inoculated primary human BMVEC with high titer macrophage-tropic HIV-1 or cocultured them with virus-infected monocytes. In both instances, BMVEC failed to demonstrate productive viral replication. Cell to cell contact between monocytes and microvascular endothelium resulted in E-selectin expression on BMVEC. BMVEC. cocultured with LPS-activated HIV-infected monocytes expressed even higher levels of E-selectin and vascular cell adhesion molecule-1 (VCAM-1). Transwell assays supported a role of soluble factors, from virus-infected monocytes, for the induction of adhesion molecules on BMVEC. To verify the in vivo relevance of these findings, levels of adhesion molecules were compared with those of proinflammatory cytokines and HIV-1 gene products in brain tissue of AIDS patients with or without encephalitis and HIV-seronegative controls. E-Selectin, and to a lesser degree VCAM-1, paralleled the levels of HIV-1 gene products and proinflammatory cytokines in brain tissue of subjects with encephalitis. Most importantly, an association between macrophage infiltration and increased endothelial cell adhesion molecules was observed in encephalitic brains. Monocyte binding to encephalitic brain tissue was blocked with Abs to VCAM-1 and E-selectin. These data, taken together, suggest that HIV entry into brain is, in part, a consequence of the ability of virus-infected and immune-activated monocytes to induce adhesion molecules on brain endothelium.     

Growth hormone axis in cushing''s syndrome

This article reviews some recent studies on alcohol preference, dependence, metabolism and pharmacokinetics which were mainly carried out in our department. The inbred strains of mice with genetically different alcohol drinking behavior and alcohol animal model treated with the neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine, are useful for a behavioral and pharmacological approach to evaluate the contribution of specific neural systems to alcohol, drug dependence mechanism and alcohol drinking behavior. The relations between alcohol preference and some physiological conditions are reviewed. On the drug-alcohol interaction, some drugs containing the chemical group = CHONO2, antimony and methamphetamine are addressed. This article also deals with recent topics in the pharmacokinetics and pharmacodynamics of alcohol. The dose-dependency of the alcohol elimination rate, the first-pass metabolism during alcohol drinking, and the pharmacodynamic model for describing pulse rate reaction to plasma acetaldehyde are discussed.