Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.     

Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Chromosomal Location of a Gene Defining Nicotinamide Deamidase in Escherichia coli

A gene specifying the enzyme nicotinamide deamidase has been mapped, by the interrupted mating technique, at about 33 min on the chromosome of Escherichia coli K-12.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

Suppressors of a Lin-12 Hypomorph Define Genes That Interact with Both Lin-12 and Glp-1 in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor which mediates cell-cell interactions required to specify certain cell fates. Reversion of the egg-laying defective phenotype caused by a hypomorphic lin-12 allele identified rare extragenic suppressor mutations in five genes, sel-1, sel-9, sel-10, sel-11 and sel(ar40) (sel = suppressor and/or enhancer of lin-12). Mutations in each of these sel genes suppress defects associated with reduced lin-12 activity, and enhance at least one defect associated with elevated lin-12 activity. None of the sel mutations cause any obvious phenotype in a wild-type background. Gene dosage experiments suggest that sel-1 and sel(ar40) mutations are reduction-of-function mutations, while sel-9 and sel-11 mutations are gain-of-function mutations. sel-1, sel-9, sel-11 and sel(ar40) mutations do not suppress amorphic lin-12 alleles, while sel-10 mutations are able to bypass partially the requirement for lin-12 activity in at least one cell fate decision. sel-1, sel-9, sel-10, sel-11 and sel(ar40) mutations are also able to suppress the maternal-effect lethality caused by a partial loss-of-function allele of glp-1, a gene that is both structurally and functionally related to lin-12. These sel genes may therefore function in both lin-12 and glp-1 mediated cell fate decisions.     

Relevance of Non-ceruloplasmin Copper to Oxidative Stress in Patients with Hepatocellular Carcinoma

Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.