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.     

JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.     

Clinical response to chemotherapy in locally advanced breast cancer was not associated with several polymorphisms in detoxification enzymes and DNA repair genes

The main aim of the present study was to investigate the association between several genetic polymorphisms (in glutathione S-transferase members and DNA repair genes) and clinical response to chemotherapy in locally advanced breast cancer. A sequential series of 101 patients were prospectively included in this study. Clinical assessment of treatment was accomplished by comparing initial tumor size with preoperative tumor size using revised RECIST guideline (version 1.1). Clinical response was regarded as a response or no response. There was no difference between non-responders and responders for the prevalence of genotypes of the study polymorphisms.     

Expression and characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> derived hepatitis C virus ARFP/F protein

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called “ARFP/F”, or “core+1,” protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11–161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the lb-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.     

Evaluation of MCM-2 expression in TMA cervical specimens

Background

Minichromosome maintenance proteins (MCM) are highly expressed in actively replicating cells. The need for biological markers for cervical carcinoma and its precursor lesions is emerging. Our main aim was to determine the immunohistochemical expression of MCM-2 in HIV-positive and -negative dysplastic cervical specimens.

Methods

Immunohistochemical analysis of MCM-2 was performed in a total of 352 cervical TMA specimens of normal control, low-grade CIN, high-grade CIN and invasive tumor. 38 specimens were from HIV-positive women. A receiver operating characteristic (ROC) curve was constructed to determine the best cutoff to diagnose high-grade CIN and invasive cervical cancer.

Results

In the progression from normal epithelium to high-grade CIN and invasive tumor we found significant differences in the MCM-2 expression (p<0.05). Based on the ROC curve of 80% with an area under the curve (AUC) of 0.78, expression of MCM-2 to diagnose high-grade CIN and invasive tumor resulted in sensitivity of 81%, specificity of 66%, a positive predictive value (PPV) of 86% and a negative predictive value (NPV) of 57%. HIV-positive cervices revealed a decreasing expression of MCM-2 in both LGCIN and HGCIN compared with HIV-negative specimens (p<0.0001).

Conclusions

The present study suggests that immunohistochemical MCM-2 may not be a promising biomarker for diagnosing high-grade CIN and invasive cancer.     

Tat–Dependent Translocation of an F420–Binding Protein of Mycobacterium tuberculosis

F₄₂₀ is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F₄₂₀ has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F₄₂₀–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F₄₂₀ during expression experiments in M. smegmatis. We also show that the Rv0132c–F₄₂₀ complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F₄₂₀–binding protein being a substrate for the Tat pathway and of the presence of F₄₂₀ outside of the cytosol in any F₄₂₀–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F₄₂₀ biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.     

Glutathione mediated reductive activation and mitochondrial dysfunction play key roles in lithium induced oxidative stress and cytotoxicity in liver

Lithium preparations are commonly used drug in treating mental disorders and bipolar diseases, but metal's cytotoxic mechanisms have not yet been completely understood. In this study, we investigated the cytotoxic mechanisms of lithium in freshly isolated rat hepatocytes. Lithium cytotoxicity were associated with reactive oxygen species (ROS) formation and collapse of mitochondrial membrane potential and cytochrome c release into the hepatocyte cytosol. All of the mentioned lithium-induced cytotoxicity markers were significantly (P?相似文献