Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Activation of proMMP-2 by U46619 occurs via involvement of p38MAPK-NFκB-MT1MMP signaling pathway in pulmonary artery smooth muscle cells

We investigated the mechanism by which TxA₂ mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p³⁸MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p³⁸MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKβ siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p³⁸MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.     

Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein.

In this paper, we report the cloning and analysis of a cDNA encoding a protein of M(r) congruent to 47,000 (p47), which is localized to the nucleus of rat hepatocytes. The cDNA showed 37% overall sequence identity with a mouse translation initiation factor, eIF-4A, which belongs to a family of putative ATP-dependent RNA helicases. We raised polyclonal antibodies against the fusion protein and by indirect immunofluorescence on primary cultures of hepatocytes have demonstrated that p47 is located in the nucleus. Although only approximately 27% of hepatocytes showed this nuclear staining, most of the nuclei in proliferating transformed cell lines such as 3T3, PtK-1, and Hela were fluorescently labeled. Studies on serum-starved cells in culture indicated that p47 was expressed in a cell cycle-dependent manner. Northern analyses demonstrated that the levels of p47 mRNA were high in fetal liver and dropped significantly after birth to low levels in adult liver. Our data suggest that p47 is developmentally regulated in rat liver at the mRNA level.     

Concise synthesis of the pentasaccharide O-antigen of Escherichia coli O83:K24:H31 present in the Colinfant vaccine

A block synthetic approach is presented for the synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the “Colifant” vaccine. The target pentasaccharide has been synthesized by coupling a disaccharide with a trisaccharide in excellent yield. Yields are quite satisfactory in all intermediate steps. A concise synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the COLINFANT vaccine is presented. The target pentasaccharide has been synthesized following a block synthetic strategy by coupling a disaccharide with a trisaccharide in excellent yield.     

Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung

Background

The variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB.

Methods/Principal Findings

A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log₁₀, p<0.01) and spleen (2.36 log₁₀, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture.

Conclusions/Significance

These results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Polymorphisms in CaSR and CLDN14 Genes Associated with Increased Risk of Kidney Stone Disease in Patients from the Eastern Part of India

Kidney stone disease (KSD) is a major clinical problem imposing a large burden for both healthcare and economy globally. In India, the prevalence of kidney stone disease is rapidly increasing. This study aimed to evaluate the association between genetic defects in vitamin D receptor (VDR), calcium sensing receptor (CaSR) and claudin 14 (CLDN14) genes and kidney stone disease in patients from eastern India. We enrolled 200 consecutive kidney stone patients (age 18–60 years) (cases) and their corresponding sex and age matched 200 normal individuals (controls). To identify genetic variants responsible for KSD, we performed sequence analysis of VDR, CaSR and CLDN14 genes. Four non-synonymous (rs1801725, rs1042636, rs1801726 and rs2228570), one synonymous (rs219780) and three intronic single nucleotide polymorphisms (SNPs) (rs731236, rs219777 and rs219778) were identified. Genotype and allele frequency analysis of these SNPs revealed that, rs1801725 (Ala986Ser), rs1042636 (Arg990Gly) of CaSR gene and rs219778, rs219780 (Thr229Thr) of CLDN14 gene were significantly associated with KSD. Serum calcium levels were significantly higher in subjects carrying 986Ser allele and calcium excretion was higher in subjects bearing 990Gly allele. In conclusion, rs1801725, rs1042636, rs219778 and rs219780 SNPs were associated with kidney stone risk in patients from the eastern part of India.     

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO₂ as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO₂-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO₂ fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.