T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.     

Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.     

Synergism of Leu-Lys rich antimicrobial peptides and chloramphenicol against bacterial cells

To investigate the antibiotic activity and synergistic effect, analogues were designed to increase not only net positive charge by Lys-substitution but also hydrophobic helix region by Leu-substitution from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P)-substitution, Lys- (positions 4, 8, 14, 15) and Leu- (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed potent antibacterial activity in minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) without having hemolytic activity. In addition, P5 and chloramphenicol has potent synergistic effect against tested cell lines. As determined by propidium iodide (PI) staining, flow cytometry showed that P5 plus chloramphenicol-treated cells had higher fluorescence intensity than untreated, P5- and chloramphenicol-treated cells. The effect on plasma membrane was examined by investigating the transmembrane potential depolarizing experiments of S. aureus with P5 and chloramphenicol. The result showed that the peptide exerts its antibacterial activity by acting on the plasma membrane. Furthermore, P5 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy. Our results suggest that peptide P5 is an excellent candidate as a lead compound for the development of novel anti-infective agents and synergistic effects with conventional antibiotic agents but lack hemolytic activity.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.     

Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.     

PHENOTYPIC STOCHASTICITY PROTECTS LYTIC BACTERIOPHAGE POPULATIONS FROM EXTINCTION DURING THE BACTERIAL STATIONARY PHASE

It is generally thought that the adsorption rate of a bacteriophage correlates positively with fitness, but this view neglects that most phages rely only on exponentially growing bacteria for productive infections. Thus, phages must cope with the environmental stochasticity that is their hosts’ physiological state. If lysogeny is one alternative, it is unclear how strictly lytic phages can survive the host stationary phase. Three scenarios may explain their maintenance: (1) pseudolysogeny, (2) diversified, or (3) conservative bet hedging. To better understand how a strictly lytic phage survives the stationary phase of its host, and how phage adsorption rate impacts this survival, we challenged two strictly lytic phage λ, differing in their adsorption rates, with stationary phase Escherichia coli cells. Our results showed that, pseudolysogeny was not responsible for phage survival and that, contrary to our expectation, high adsorption rate was not more detrimental during stationary phase than low adsorption rate. Interestingly, this last observation was due to the presence of the “residual fraction” (phages exhibiting extremely low adsorption rates), protecting phage populations from extinction. Whether this cryptic phenotypic variation is an adaptation (diversified bet hedging) or merely reflecting unavoidable defects during protein synthesis remains an open question.