No abstract

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the ␤-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf and located consensus hexamers of ؊10 (CAAGAT) and ؊35 (TTCAAA), which are commonly found in 70 -dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and ␤-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on 70 , that H-NS binds to the regulatory sequence of lpfA and "silences" the transcription of lpf, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a serious food-borne pathogen causing diarrhea that is often bloody and accompanied by severe abdominal cramps and can result in a life-threatening condition known as the hemolyticuremic syndrome (reviewed in reference 33). The organism can be found living in the intestines of healthy cattle, and eating contaminated meat, especially ground beef, has resulted in multiple outbreaks worldwide (23). Recent E. coli O157:H7 outbreaks have drawn attention to food-borne illnesses, and though official sources were saying that the overall number of cases is on the decline in the United States, consumption of produce, particularly leafy vegetables, is becoming increasingly associated with human infections. This alternate source of infection has set up new challenges for the scientific community in trying to identify novel determinants and regulatory mechanisms implicated in the colonization, survival, and/or pathogenic processes (9,10,40).During the infectious process, EHEC adheres to the intestinal epithelium, where it produces Shiga toxins responsible for the hemorrhagic symptoms. Adhesion of E. coli O157:H7 t...

Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates the expression of Galectin-9 has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of this study were: (a) to identify the mRNA variants of LGALS9, (b) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene, and (c) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. Chromatin immunoprecipitation analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation, but not promoter CpG methylation, may be involved in the transcriptional regulation of the LGALS9 gene. Cervical cancer (CC) is the second most lethal cancer among women in undeveloped countries [1]. The aetiological agent for this cancer is human papillomavirus (HPV), and persistent infection with some genotypes of HPV has been associated with the development of CC [2].

ST3GAL4 gene expression is altered in different cancer types, including cervical cancer. Several mRNA transcripts have been reported for this gene; however, their expression levels in cervical cancer have not been analyzed. ST3GAL4 encodes for β‑galactosidase α‑2,3‑sialyltransferase 4, involved in the biosynthesis of the tumour antigens sLe(x) and sulfo‑sLe(x). The present study evaluated the presence of three mRNA variants (V1, V2 and V3) in cervical cancer cell lines, detecting the three variants. Additionally, the expression level of the V1 transcript of the ST3GAL4 gene was determined by reverse transcription‑quantitative polymerase chain reaction in cervical cell lines and in normal, premalignant and cervical cancer tissue. The V1 transcript of the ST3GAL4 demonstrated significant decreased expression in premalignant and malignant cervical tissues. The results suggested that deregulation of this gene could occur prior to the presence of cancer and demonstrated the importance of evaluating the expression level of V1, and its association with disease progression.

The level of beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) mRNA, encoded by the gene siat1, is increased in malignant tissues. Expression is regulated by different promoters - P1, P2 and P3 - generating three mRNA isoforms H, X and YZ. In cervical cancer tissue the mRNA isoform H, which results from P1 promoter activity, is increased. To study the regulation of P1 promoter, different constructs from P1 promoter were evaluated by luciferase assays in cervical and hepatic cell lines. Deletion of a fragment of 1048 bp (-89 to +24 bp) increased 5- and 3-fold the promoter activity in C33A and HepG2 cell lines, respectively. The minimal region with promoter activity was a 37 bp fragment in C33A cells. The activity of this region does not require the presence of an initiator sequence. In HepG2 cells the minimal promoter activity was detected in the 66 bp fragment. Sp1 (-32) mutation increased the promoter activity only in HepG2 cells. HNF1 mutation decreased promoter activity in HepG2 cell line but not in C33A cells. We identified a large region that plays a negative regulation role. The regulation of promoter activity is cell type specific. Our study provides new insights into the complex transcriptional regulation of siat1 gene.