Expression of HIV-1nefin yeast causes membrane perturbation and release of the myristylated Nef protein

The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.     

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Background

The topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α.

Methodology/Principal Findings

We show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.

Conclusions/Significance

The discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.     

Morpholine-based RGD-cyclopentapeptides as αvβ3/αvβ5 integrin ligands: Role of configuration towards receptor binding affinity

Two c[RGDfX] cyclopeptides, having either l- or d-morpholine-3-COOH (Mor) as the X amino acid were developed as ligands for α_vβ₃/α_vβ₅ integrins. Biological assays showed only d-Mor-containing cyclopentapeptide capable to bind α_vβ₃ integrin with a low nanomolar affinity according to a two-site model, thus revealing a connection between the configuration of Mor and the preferred binding to α_vβ₃ integrin. Conformational analysis showed different structural preferences for the two peptides induced by the two enantiomeric cyclic amino acids, suggesting a role of the stereochemistry of Mor on the overall peptide conformation and on the presentation of the pharmacophoric Arg and Asp side chains.     

Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor

In the differentiating eubacterium Streptomyces coelicolor , nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon the growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the levels of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.     

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley-Liss, Inc.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5′ untranslated region. “Uroporphyric” mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype chracteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12 The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the β-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.     

An improved ontological representation of dendritic cells as a paradigm for all cell types

Background  

Recent increases in the volume and diversity of life science data and information and an increasing emphasis on data sharing and interoperability have resulted in the creation of a large number of biological ontologies, including the Cell Ontology (CL), designed to provide a standardized representation of cell types for data annotation. Ontologies have been shown to have significant benefits for computational analyses of large data sets and for automated reasoning applications, leading to organized attempts to improve the structure and formal rigor of ontologies to better support computation. Currently, the CL employs multiple is_a relations, defining cell types in terms of histological, functional, and lineage properties, and the majority of definitions are written with sufficient generality to hold across multiple species. This approach limits the CL's utility for computation and for cross-species data integration.