Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.     

India takes an open source approach to drug discovery

Open source software may have been around for 17 years, but using an open source model to speed up drug discovery is a relatively new idea. This month, India is launching a new open source initiative for developing drugs to treat diseases such as tuberculosis, malaria, and HIV.     

Cultivable Bacterial Diversity of Alkaline Lonar Lake, India

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 10²–10⁶ cfu g⁻¹ and 10²–10⁴ cfu ml⁻¹, respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to γ-Proteobacteria, Alcaligenes to β-Proteobacteria and Paracoccus to α-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.     

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.     

A novel role for villin in intestinal epithelial cell survival and homeostasis

Apoptosis is a key regulator for the normal turnover of the intestinal mucosa, and abnormalities associated with this function have been linked to inflammatory bowel disease and colorectal cancer. Despite this, little is known about the mechanism(s) mediating intestinal epithelial cell apoptosis. Villin is an actin regulatory protein that is expressed in every cell of the intestinal epithelium as well as in exocrine glands associated with the gastrointestinal tract. In this study we demonstrate for the first time that villin is an epithelial cell-specific anti-apoptotic protein. Absence of villin predisposes mice to dextran sodium sulfate-induced colitis by promoting apoptosis. To better understand the cellular and molecular mechanisms of the anti-apoptotic function of villin, we overexpressed villin in the Madin-Darby canine kidney Tet-Off epithelial cell line to demonstrate that expression of villin protects cells from apoptosis by maintaining mitochondrial integrity thus inhibiting the activation of caspase-9 and caspase-3. Furthermore, we report that the anti-apoptotic response of villin depends on activation of the pro-survival proteins, phosphatidylinositol 3-kinase and phosphorylated Akt. The results of our studies shed new light on the previously unrecognized function of villin in the regulation of apoptosis in the gastrointestinal epithelium.     

Function, expression, and characterization of the serotonin transporter in the native human intestine

The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum > duodenum > jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the human ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band ( approximately 70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [(3)H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both Na(+) and Cl(-); 2) inhibited ( approximately 50%) by the neuronal SERT inhibitor, fluoxetine (10 microM) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells.     

Potent Upregulation of Glutathione and NAD(P)H:Quinone Oxidoreductase 1 by Alpha-lipoic Acid in Human Neuroblastoma SH-SY5Y Cells: Protection Against Neurotoxicant-elicited Cytotoxicity

Alpha-lipoic acid (LA) has recently been reported to afford protective effects in neurodegenerative disorders. However, the mechanisms underlying LA-mediated neuroprotection remain to be investigated. This study was undertaken to determine whether LA treatment could increase endogenous antioxidants and phase 2 enzymes in cultured human neuroblastoma SH-SY5Y cells, and whether such increased cellular defenses could afford protection against cytotoxicity induced by neurotoxicants. Incubation of SH-SY5Y cells with micromolar concentrations of LA for 24 h resulted in a significant increase in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQQ1) in a concentration-dependent fashion. Treatment of the cells with LA also led to an increased mRNA expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. To determine the protective effects of the LA-induced cellular defenses on neurotoxicant-elicitedl cell injury, SH-SY5Y cells were pretreated with LA for 24 h and then exposed to acrolein, 4-hydroxy-2-nonenal (HNE), H₂O₂ and the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1). We observed that LA pretreatment of SH-SY5Y cells led to a marked protection against acrolein, HNE, H₂O₂ and SIN-1-mediated cytotoxicity, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Taken together, this study demonstrates for the first time that LA can induce GSH and NQO1 in cultured human neuroblastoma cells and LA-upregulated cellular defenses are accompanied by a markedly increased resistance to cytotoxicity induced by various neurotoxicants. The results of this study may have important implications for the neuroprotective effects of LA.     

The applicability of hns and fimA primers for detecting Salmonella in bioaerosols associated with animal and municipal wastes

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.     

Suppression of atrial natriuretic peptide/natriuretic peptide receptor-A-mediated signaling upregulates angiotensin-II-induced collagen synthesis in adult cardiac fibroblasts

Cardiac hormone atrial natriuretic peptide (ANP) and its receptor natriuretic peptide receptor-A (NPR-A) system acts as an intrinsic negative regulator of abnormal extracellular matrix (ECM) remodeling in the heart. However, the underlying mechanism by which ANP/NPR-A system opposes the ECM remodeling in the diseased heart is not well understood. Here, we investigated the anti-fibrotic mechanism of ANP/NPR-A in fibrotic agonist Angiotensin- II (ANG II)-treated adult cardiac fibroblast (CF) cells. Normal and NPR-A-suppressed adult CF cells were treated with ANG II (10^?7 M) in the presence and absence of ANP (10^?8 M) for 24 h. Total collagen concentration, activity and expression of MMP-2 and MMP-9, and nuclear translocation of Nuclear factor-kappaB (NF-κB-p50) were studied. NPR-A-suppressed adult CF cells exhibited a more pronounced increase in collagen production, ROS generation, and NF-κB-p50 nuclear translocation as compared to adult CF cells treated with agonist alone. ANP co-treatment significantly reverses the agonist-induced above changes in normal adult CF cells, while it failed to reverse the agonist-induced collagen synthesis in the NPR-A-suppressed adult CF cells. The cGMP analog (8-bromo-cGMP) treatment significantly attenuated the agonist-induced collagen synthesis both in normal and NPR-A-suppressed adult cells. The results of this study suggest that ANP/NPR-A signaling system antagonizes the agonist-induced collagen synthesis via suppressing the activities of MMP-2, MMP-9, ROS generation, and NF-κB nuclear translocation mechanism.