UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Crocidolite asbestos inhibits pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human lung epithelial cells

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.     

A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli.

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

Adverse environmental conditions influence age-related innate immune responsiveness

Background-  

The innate immune system plays an important role in the recognition and induction of protective responses against infectious pathogens, whilst there is increasing evidence for a role in mediating chronic inflammatory diseases at older age. Despite indications that environmental conditions can influence the senescence process of the adaptive immune system, it is not known whether the same holds true for the innate immune system. Therefore we studied whether age-related innate immune responses are similar or differ between populations living under very diverse environmental conditions.     

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

A GWAS assessment of the contribution of genomic imprinting to the variation of body mass index in mice

Background

Genomic imprinting is an epigenetic mechanism that can lead to differential gene expression depending on the parent-of-origin of a received allele. While most studies on imprinting address its underlying molecular mechanisms or attempt at discovering genomic regions that might be subject to imprinting, few have focused on the amount of phenotypic variation contributed by such epigenetic process. In this report, we give a brief review of a one-locus imprinting model in a quantitative genetics framework, and provide a decomposition of the genetic variance according to this model. Analytical deductions from the proposed imprinting model indicated a non-negligible contribution of imprinting to genetic variation of complex traits. Also, we performed a whole-genome scan analysis on mouse body mass index (BMI) aiming at revealing potential consequences when existing imprinting effects are ignored in genetic analysis.

Results

10,021 SNP markers were used to perform a whole-genome single marker regression on mouse BMI using an additive and an imprinting model. Markers significant for imprinting indicated that BMI is subject to imprinting. Marked variance changed from 1.218 ×10⁻⁴ to 1.842 ×10⁻⁴ when imprinting was considered in the analysis, implying that one third of marked variance would be lost if existing imprinting effects were not accounted for. When both marker and pedigree information were used, estimated heritability increased from 0.176 to 0.195 when imprinting was considered.

Conclusions

When a complex trait is subject to imprinting, using an additive model that ignores this phenomenon may result in an underestimate of additive variability, potentially leading to wrong inferences about the underlying genetic architecture of that trait. This could be a possible factor explaining part of the missing heritability commonly observed in genome-wide association studies (GWAS).     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.