A human novel gene DERPC on 16q22.1 inhibits prostate tumor cell growth and its expression is decreased in prostate and renal tumors

BACKGROUND: Deletion of chromosome 16q is frequently associated with diverse tumors. Numerous studies strongly suggest the presence of one or more tumor suppressor genes on chromosome 16q22 to 16qter including the widely studied cadherin gene family. However, the specific tumor suppressor genes residing in this region need better definition and characterization. MATERIAL AND METHODS: Standard molecular biology approaches have been used to clone and characterize the DERPC cDNA and its protein product on chromosome 16q22.1. Northern blotting was used to define the expression pattern in a multiple human tissue blots. DERPC expression was examined in multi-tumor array (Clontech, CA, USA) dot blot as well as in laser capture microdissection (LCM) derived prostate cancer (CaP) specimens by quantitative RT-PCR. Western blot analysis and a fluorescent microscopy were used to characterize the molecular size and the cellular location of green fluorescent protein (GFP)-tagged DERPC fusion proteins. A colony formation assay was conducted to determine the effects of DERPC expression on tumor cell growth. RESULTS: A novel gene DERPC (Decreased Expression in Renal and Prostate Cancer) was identified and characterized. DERPC encoded a strong basic, proline- and glycine-rich nuclear protein. DERPC was ubiquitously expressed, with abundant expression in kidney, skeletal muscle, testis, liver, ovary, and heart and moderate expression in prostate. DERPC expression was reduced in renal (67%) and prostate tumors (33%). Expression of DERPC has inhibitory potential on CaP cell growth. Further, overexpression of DERPC in LNCaP cells caused alterations of nuclear morphology. CONCLUSION: This study suggests that decreased expression of DERPC may be implicated in tumorigenesis of renal and CaPs.     

Synthesis and energy transfer efficiency of FRET terminators derived from different linkers

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5'-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.     

Effect of aerosolized acetylcholine on bronchial blood flow

We studiedthe effects of aerosolized as well as intravenous infusion ofacetylcholine on bronchial blood flow in six anesthetized sheep.Intravenous infusion of acetylcholine, at a dose of 2 µg/kg, increased bronchial blood flow from 45 ± 15 (SE) to 74 ± 30 ml/min, and vascular conductance increased by 76 ± 22%. In contrast, aerosolized acetylcholine at doses of 2 and 20 µg/kg decreased bronchial vascular conductance by ~10%. At anaerosolized dose of 200 µg/kg, the bronchial vascular conductanceincreased by ~15%, and there was no further increase in conductancewhen the aerosolized dose was increased to 2,000 µg/kg. Pretreatmentof animals with a nitric oxide synthase inhibitor,N-nitro-L-argininemethyl ester hydrochloride, partially blocked the vasodilatory effectsof intravenous acetylcholine and completely blocked the vasodilatoryeffects of high-dose aerosolized acetylcholine. These data suggest thataerosolized acetylcholine does not readily penetrate the vascular wallof bronchial circulatory system and, therefore, has minimalvasodilatory effects on the bronchial vasculature.

     

Gradual pediocin PA-1 resistance in <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> confers cross-protection to diverse pore-forming cationic antimicrobial peptides displaying changes in cell wall and mannose PTS expression

Due to innate and acquired resistance in Enterococcus faecalis against most antibiotics, identification of new alternatives has increased interest in diverse populations of potent cationic antimicrobial peptides (CAMPs) for treatment and natural food biopreservation. The CAMPs, after crossing the cell wall to the periplasmic space, kill their target strain by forming pores in the cell membrane. However, reports of resistance against these CAMPs necessitated the understanding of step(s) interfered with while acquiring this resistance, for designing effective CAMP analogs. In this direction, we selected stable and gradual dose-dependent pediocin PA-1 single exposure resistant (Ped^r) mutants of E. faecalis, which conferred cross-protection to diverse CAMPs, viz., HNP-1, nisin and alamethicin but not to polymyxin B, lysozyme and vancomycin. With these Ped^r mutants of E. faecalis there was: a gradual neutralization in cell wall surface charge involving D-alanylation of wall teichoic acids (WTA) and lipoteichoic acids (LTA), increase in cell-surface hydrophobicity, increased cell aggregation and biofilm formation and ultra-structural changes in the cell wall, and a reduction of periplasmic space. In addition, a gradual decrease in expression of mannose PTS two (mpt) operon was also observed with distinct changes in growth rate achieving the same biomass production during the stationary phase. These results show that resistance to these CAMPs is not due to mpt directly acting as a docking molecule but due to changes in the cell wall, which increased the permeability barrier to CAMPs diffusion to reach the periplasmic space.     

Genetic Diversity in Lens Species Revealed by EST and Genomic Simple Sequence Repeat Analysis

Low productivity of pilosae type lentils grown in South Asia is attributed to narrow genetic base of the released cultivars which results in susceptibility to biotic and abiotic stresses. For enhancement of productivity and production, broadening of genetic base is essentially required. The genetic base of released cultivars can be broadened by using diverse types including bold seeded and early maturing lentils from Mediterranean region and related wild species. Genetic diversity in eighty six accessions of three species of genus Lens was assessed based on twelve genomic and thirty one EST-SSR markers. The evaluated set of genotypes included diverse lentil varieties and advanced breeding lines from Indian programme, two early maturing ICARDA lines and five related wild subspecies/species endemic to the Mediterranean region. Genomic SSRs exhibited higher polymorphism in comparison to EST SSRs. GLLC 598 produced 5 alleles with highest gene diversity value of 0.80. Among the studied subspecies/species 43 SSRs detected maximum number of alleles in L. orientalis. Based on Nei’s genetic distance cultivated lentil L. culinaris subsp. culinaris was found to be close to its wild progenitor L. culinaris subsp. orientalis. The Prichard’s structure of 86 genotypes distinguished different subspecies/species. Higher variability was recorded among individuals within population than among populations.     

Modeling-Enabled Characterization of Novel NLRX1 Ligands

Nucleotide-binding domain and leucine-rich repeat containing (NLR) family are intracellular sentinels of cytosolic homeostasis that orchestrate immune and inflammatory responses in infectious and immune-mediated diseases. NLRX1 is a mitochondrial-associated NOD-like receptor involved in the modulation of immune and metabolic responses. This study utilizes molecular docking approaches to investigate the structure of NLRX1 and experimentally assesses binding to naturally occurring compounds from several natural product and lipid databases. Screening of compound libraries predicts targeting of NLRX1 by conjugated trienes, polyketides, prenol lipids, sterol lipids, and coenzyme A-containing fatty acids for activating the NLRX1 pathway. The ligands of NLRX1 were identified by docking punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA) to the C-terminal fragment of the human NLRX1 (cNLRX1). Their binding and that of positive control RNA to cNLRX1 were experimentally determined by surface plasmon resonance (SPR) spectroscopy. In addition, the ligand binding sites of cNLRX1 were predicted in silico and validated experimentally. Target mutagenesis studies demonstrate that mutation of 4 critical residues ASP677, PHE680, PHE681, and GLU684 to alanine resulted in diminished affinity of PUA, ESA, and DHA to NLRX1. Consistent with the regulatory actions of NLRX1 on the NF-κB pathway, treatment of bone marrow derived macrophages (BMDM)s with PUA and DHA suppressed NF-κB activity in a NLRX1 dependent mechanism. In addition, a series of pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the regulatory function of PUA on colitis is NLRX1 dependent. Thus, we identified novel small molecules that bind to NLRX1 and exert anti-inflammatory actions.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who control viremia to levels below the limit of detection of current assays. The mechanisms involved in this control have not been fully elucidated. Several studies have demonstrated that some ES are infected with defective viruses, but it remains unclear whether others are infected with replication-competent HIV-1. To answer this question, we used a sensitive coculture assay in an attempt to isolate replication-competent virus from a cohort of 10 ES. We successfully cultured six replication-competent isolates from 4 of the 10 ES. The frequency of latently infected cells in these patients was more than a log lower than that seen in patients on highly active antiretroviral therapy with undetectable viral loads. Full-length sequencing of all six isolates revealed no large deletions in any of the genes. A few mutations and small insertions and deletions were found in some isolates, but phenotypic analysis of the affected genes suggested that their function remained intact. Furthermore, all six isolates replicated as well as standard laboratory strains in vitro. The results suggest that some ES are infected with HIV-1 isolates that are fully replication competent and that long-term immunologic control of replication-competent HIV-1 is possible.