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HEp-2 Cells (hep-2 + cell)
Selected AbstractsAggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2007Sérgio P.D. Rocha Abstract Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK2 cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus -like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients. [source] Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patientsFEMS MICROBIOLOGY LETTERS, Issue 1 2000Sunao Iyoda Abstract We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC. [source] Novel Brush Polymers with Phosphorylcholine Bristle Ends: Synthesis, Structure, Properties, and BiocompatibilityADVANCED FUNCTIONAL MATERIALS, Issue 10 2009Gahee Kim Abstract New brush polymers with various numbers of bristle ends incorporating phosphorylcholine (PC) moieties are synthesized. The polymers are thermally stable up to 175,°C and form good-quality films with conventional spin-, roll-, and dip-coating, and subsequent drying processes. Interestingly, all these brush polymers, as a PC-containing polymer, demonstrate a stable molecular multi-bilayer structure in thin films that arise due to the efficient self-assembly of the bristles for temperatures <55,°C and PC-rich surfaces, and therefore successfully mimic natural cell-membrane surfaces. These brush-polymer films exhibit excellent water wettability and water sorption whilst retaining the remarkable molecular multi-bilayer structure, and thus have hydrophilic surfaces. These novel multi-bilayer structured films repel fibrinogen molecules and platelets from their surfaces but also have bactericidal effects on bacteria. Moreover, the brush-polymer films are found to provide comfortable surface environments for the successful anchoring and growth of HEp-2 cells, and to exhibit excellent biocompatibility in mice. These newly developed brush polymers are suitable for use in biomedical applications including medical devices and biosensors that require biocompatibility and the reduced possibility of post-operative infection. [source] Different apoptosis ratios and gene expressions in two human cell lines after sevoflurane anaesthesiaACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2009S. KVOLIK Background: The aim of this study was to determine the effect of a single exposure of carcinoma cells (Caco-2 and HEp-2) to an anaesthetic gas mixture containing sevoflurane 3%, applied for a period of either 1 or 2 h, on the induction of apoptosis, propapototic gene expression and sphingomyelinase activity. Methods: Apoptosis was determined by flow cytometry. p53, caspase 3 and CYP2E1 gene expression was determined using reverse transcriptase polymerase chain reaction. Activities of acid (aSMase) and neutral sphingomyelinases (nSMase) were measured using methyl- 14C sphingomyeline, and for de novo ceramide and lipid synthesis [3H] palmitic acid was used. All results were compared with controls and analysed by Mann,Whitney and Kruskal,Wallis tests. Results: In the treated Caco-2 cells, the apoptotic ratio increased 24 h after anaesthesia (16.9%; P=0.04). The expression of both p53 and caspase-3 genes increased in Caco-2 and decreased in HEp-2 cells. The CYP2E1 gene expression was observed only in the Caco-2 cells. In control cells, the catalytic activity of aSMase was 2.3 times higher than that of nSMase activity. Decreased aSMase and nSMase activities were observed in Caco-2 cells 24 h after exposition. aSMase activity was halved (54.2%; P=0.06) in HEp-2 cells 24 h after anaesthesia. De novo ceramide synthesis correlated with SMase activity in Caco-2 cells. Conclusion: Sevoflurane anaesthesia induces late apoptosis in the colonic and laryngeal cancer cells investigated. Although the results obtained may indicate that an anaesthetic gas mixture containing sevoflurane induces p53-dependent apoptosis in the Caco-2 cells, the mechanism of apoptosis induction is unclear and remains to be elucidated. [source] Antibodies to SS-A/Ro-52kD and centromere in autoimmune liver disease: a clue to diagnosis and prognosis of primary biliary cirrhosisALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2007A. GRANITO Summary Background Primary biliary cirrhosis (PBC) may be associated with various rheumatological disorders. Aim To investigate the frequency and significance of ,rheumatological' antinuclear antibodies in the field of autoimmune chronic liver disease, with special regard to PBC. Methods We studied 105 patients with PBC, 162 autoimmune liver disease controls (type 1 and 2 autoimmune hepatitis, primary sclerosing cholangitis), 30 systemic lupus erythematosus and 50 blood donors. Sera were tested for the presence of antibodies to extractable nuclear antigens (anti-ENA) by counterimmunoelectrophoresis, enzyme-linked and immunoblot (IB) assay, and for the presence of anti-centromere antibodies (ACA) by indirect immunofluorescence on HEp-2 cells and IB. Results The overall prevalence of IB-detected anti-ENA in PBC (30%) was higher than in type 1 autoimmune hepatitis (2.5%, P < 0.0001), type 2 autoimmune hepatitis (0%, P < 0.0001) and primary sclerosing cholangitis (11.5%, P = 0.006) and lower than in systemic lupus erythematosus (53%, P = 0.03). The most frequent anti-ENA reactivity in PBC was anti-SSA/Ro-52kD (28%). ACA were detected by IB in 21% PBC patients and never in the other subjects (P < 0.0001). Anti-SS-A/Ro/52kD positive PBC patients had at the time of diagnosis a more advanced histological stage (P = 0.01) and higher serum levels of bilirubin (P = 0.01) and IgM (P = 0.03) compared with negative ones. Conclusions In the autoimmune liver disease setting, anti-SS-A/Ro-52kD and ACA have a high specificity for PBC and can thus be of diagnostic relevance in anti-mitochondrial antibodies negative cases. If confirmed in further studies with adequate follow-up, anti-SS-A/Ro-52kD antibodies might identify PBC patients with a more advanced and active disease. [source] Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and ThailandMICROBIOLOGY AND IMMUNOLOGY, Issue 4 2010Mariko Iida ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae - and bfpA - positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n= 27) and Thailand (n= 26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan. [source] Differential binding to and biofilm formation on, HEp-2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene clusterMOLECULAR MICROBIOLOGY, Issue 5 2002Jennifer D. Boddicker Summary Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process. [source] Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp-2 cells via reactive oxygen species productionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2010Jae-Sung Kim Abstract The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp-2 cells with fractionated radiation. These RR-HEp-2 cells and isolated clones displayed more radioresistant and anti-apoptotic phenotypes than parental HEp-2 cells after radiation. Characteristics of RR-Hep-2 cell lines were confirmed by upregulation of radioresistance-related genes, such as epidermal growth factor receptor, Hsp90, and Bcl-xl. Subsequently, we examined proteome changes between HEp-2 and RR-HEp-2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR-HEp-2 cells, compared with non-irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid-94 or small interfering RNA led to radioresistance in HEp-2 cells by suppressing the radiation-induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR-HEp-2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells. [source] Proteomic analysis of cells in the early stages of herpes simplex virus type-1 infection reveals widespread changes in the host cell proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2009Robin Antrobus Abstract During infection by herpes simplex virus type-1 (HSV-1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV-1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV-1 replication, 2-DE and LC-MS/MS were used to identify cellular proteome changes at 6,h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock- and HSV-1-infected HEp-2 cells revealed a total of 103 protein spot changes. Of these, 63 were up-regulated and 40 down-regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV-1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV-1 infection. [source] Antinuclear antibodies following infliximab treatment in patients with rheumatoid arthritis or spondylarthropathyARTHRITIS & RHEUMATISM, Issue 4 2003Leen De Rycke Objective To investigate the effect of infliximab treatment on antinuclear antibodies (ANAs), anti,double-stranded DNA (anti-dsDNA), antinucleosome, antihistone, and anti,extractable nuclear antigen (anti-ENA) antibodies in rheumatoid arthritis (RA) and spondylarthropathy (SpA) patients. Methods Sera from 62 RA and 35 SpA patients treated with infliximab were tested at baseline and week 30 (RA group) or week 34 (SpA group). ANAs were tested by indirect immunofluorescence (IIF) on HEp-2 cells. Anti-dsDNA antibodies were detected by IIF on Crithidia luciliae and by enzyme-linked immunosorbent assay (ELISA) and were further isotyped with ,, ,, and , chain,specific conjugates at various time points. Antinucleosome antibodies were tested by ELISA. Antihistone and anti-ENA antibodies were detected by line immunoassay. Results Initially, 32 of 62 RA patients and 6 of 35 SpA patients tested positive for ANAs. After infliximab treatment, these numbers shifted to 51 of 62 (P < 0.001) and 31 of 35 (P < 0.001), respectively. At baseline, none of the RA or SpA patients had anti-dsDNA antibodies. After infliximab treatment, 7 RA patients (P = 0.016) and 6 SpA patients (P = 0.031) became positive for anti-dsDNA antibodies. All 7 anti-dsDNA,positive RA patients had IgM and IgA anti-dsDNA antibodies. Three of the 6 anti-dsDNA,positive SpA patients had IgM and IgA anti-dsDNA antibodies, and 2 had IgM anti-dsDNA antibodies alone. In both diseases, the IgM anti-dsDNA antibodies appeared before the IgA anti-dsDNA antibodies. During the observation period, no IgG anti-dsDNA antibodies or lupus symptoms were observed. The development of antinucleosome, antihistone, or anti-ENA antibodies following infliximab treatment was observed in some patients, but the numbers were not statistically significant. Conclusion Infliximab treatment may induce ANAs, and especially IgM and IgA anti-dsDNA antibodies, in RA and SpA patients. However, no anti-dsDNA IgG antibodies or lupus symptoms were observed during the period of observation in this study, and the development of antinucleosome, antihistone, or anti-ENA antibodies was not statistically significant. These observations do not exclude potential induction of clinically significant lupus in the long term, and further followup is therefore mandatory. [source] Temporal arteritis and Chlamydia pneumoniae: Failure to detect the organism by polymerase chain reaction in ninety cases and ninety controlsARTHRITIS & RHEUMATISM, Issue 4 2002Michael J. Regan Objective To examine the reported correlation between the presence of Chlamydia pneumoniae in temporal artery biopsy specimens and the diagnosis of temporal arteritis (TA). Methods Among 90 possible cases of TA identified at our institution between 1968 and 2000, 79 of the positive biopsy specimens (88%) demonstrated giant cells and the other 11 cases (12%) had other histopathologic features compatible with TA; by chart review, all 90 patients were confirmed to have met the American College of Rheumatology classification criteria for TA. Controls had negative temporal artery biopsy specimens during the same 32-year time period and their postbiopsy disease courses were not compatible with TA. Controls were matched with each case by sex, year of biopsy, and age within 10 years. The biopsy specimens from all cases and controls were reevaluated and readings were confirmed in a masked manner by an experienced eye pathologist. Polymerase chain reaction (PCR) analyses for C pneumoniae were performed on the 180 samples using 2 different sets of PCR primers (which target 2 different genes). A primer set targeting the ompA gene (CP1-CP2/CPC-CPD) was used to perform a nested PCR, followed by confirmation of the findings with primers targeting the 16S ribosomal RNA (rRNA) gene (Cpn90/Cpn91) in a touchdown-enzyme time-release PCR. We used positive and negative controls, as well as controls made from infected and noninfected HEp-2 cells, suspended in a formalin-fixed, paraffin-embedded matrix. Results Seventy-six percent of the 180 cases and controls were women. The mean age of the cases was 72.0 years (range 53,90), and that of the controls was 70.4 years (range 51,86). Eighty percent of the control samples were obtained by temporal artery biopsy performed within 1 year of the biopsies performed on the matched cases. Using the CP1-CP2/CPC-CPD primer set, only 1 TA case sample (1% of all case samples) was positive for the ompA gene. One control sample was also positive using these primers. With the Cpn90/Cpn91 primers, none of the cases and none of the controls were positive for the 16S rRNA gene. Conclusion The results of this study using sensitive and specific PCR analyses do not support a role for C pneumoniae in the pathogenesis of TA. [source] Adherence and invasion of Bacteroidales isolated from the human intestinal tractCLINICAL MICROBIOLOGY AND INFECTION, Issue 10 2008V. Nakano Abstract Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells. [source] Cytotoxicity of doxorubicin-loaded Con A-liposomesDRUG DEVELOPMENT RESEARCH, Issue 5 2006Hercília Maria Lins Rolim Santos Abstract The present study investigated the potential of Concanavalin A lectin (Con A) conjugated to liposomes (Con A-liposomes) for targeting doxorubicin (DOX) to cells. The physicochemical properties and the cytotoxicity of DOX-loaded Con A-liposomes were evaluated. DOX-loaded Con A-liposomes were prepared by incubation of DOX-loaded liposomes with a Con A-SATA derivative. Lectin biological activity was monitored before and after conjugation by a hemagglutinating assay. The cytotoxicity of DOX-loaded Con A-liposomes was evaluated in terms of the inhibition of NCI-H299 and HEp-2 cell proliferation using the MTT method. The affinity of lectinized liposomes with these cells was thus assessed by evaluating the cytotoxic effect of the DOX released into cells. Stable DOX-loaded Con A-liposomes were obtained and their high affinity for cells was corroborated. The encapsulation of DOX into Con A-liposomes produced an inhibition of roughly 70% of Hep-2 cell proliferation and 50% of cell inhibition was verified on HCI-H292. DOX in solution was able to inhibit only 20% of cell proliferation for both cell lines. Unloaded Con A-liposomes were not cytotoxic. The encapsulation of DOX into Con A-liposomes improves drug penetration into cells, thereby enhancing its cytotoxicity, especially in Hep-2 cells. Drug Dev. Res. 67:430,437, 2006. © 2006 Wiley-Liss, Inc. [source] Inhibition of cell proliferation and glucose uptake in human laryngeal carcinoma cells by antisense oligonucleotides against glucose transporter-1HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 12 2009Shui-Hong Zhou PhD Abstract Background. Malignant cells show increased glucose uptake in vitro and in vivo, which is thought to be mediated by glucose transporters. In this study, we investigated the effect of plasmid-derived antisense RNA against the Glut-l gene on proliferation and glucose uptake in laryngeal carcinoma Hep-2 cells. Methods. The expression plasmids pcDNA3.1(+)-Glut-1 and pcDNA3.1(+)-anti Glut-1 were constructed. The MTT method was used to assess cell growth inhibition. The expression of Glut-1 mRNA and protein was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Results. After transfection, Glut-1 AS clearly inhibited glucose uptake and cell growth in Hep-2 cells, and we observed a decrease in the expression of Glut-1 mRNA and protein in Hep-2 cells. Conclusions. Glut-1 AS decreases glucose uptake and inhibits the proliferation of Hep-2 cells. © 2009 Wiley Periodicals, Inc. Head Neck, 2009 [source] In vivo investigation of CD133 as a putative marker of cancer stem cells in Hep-2 cell lineHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 1 2009Xu Dong Wei PhD Abstract Background Mounting evidence suggests that most tumors consist of a heterogeneous population of cells with a subset population that has the exclusive tumorigenic ability. They are called cancer stem cells (CSCs). CSCs can self-renew to generate additional CSCs and also differentiate to generate phenotypically diverse cancer cells with limited proliferative potential. They have been identified in a variety of tumors. In this study, we identify the marker of CSCs in the established human laryngeal tumor Hep-2 cell line in vivo. Our in vitro experiment shown as CD133, a 5-transmembrane glycoprotein expressed in Hep-2 cell line. CD133 was supposed as a candidate of CSC in laryngeal carcinoma. In this study, the expression of CD133 was detected in a Hep-2 cell line. Applying the magnetic cell sorting (MACS) technology, we reported the results of purifying CD133 positive cells from a Hep-2 cell line. Three-type cells' tumor-forming ability was examined in vivo to identify the marker of CSCs in Hep-2 cell line. Methods CD133 was selected as a putative marker of CSC in laryngeal carcinoma, Hep-2 cell lines. Flow cytometry was used to detect the expression of CD133 in the Hep-2 cell line. Immunomagnetic beads were applied to purify CD133-positive cells. CD133(+), CD133(,) tumor cells, and unsorted Hep-2 cells were injected into severe combined immune deficiency (SCID) mice individually to observe tumor-forming ability. Results Only a small proportion (3.15% ± 0.83%) of cells in the Hep-2 cell line express the CD133 marker. In comparison with CD133(,) tumor cells and unsorted cells, CD133(+) cells possess a marked capacity for tumor formation in vivo (p <.05). Conclusion CD133 is 1 of the markers for CSCs in human laryngeal tumors of the Hep-2 cell line. Work on the characterization of these cells provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting the CSC. © 2008 Wiley Periodicals, Inc. Head Neck, 2009 [source] Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitroLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2008A.A. Hugo Abstract Aims:, To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro. Methods and Results:, Strains CIDCA 133 (Lactobacillus delbrueckii subsp. lactis) and CIDCA 83114 (Lactobacillus plantarum) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells. Conclusions:, Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen. Significance and Impact of the Study:, Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC. [source] Comparative Proteomics Analysis of the Proteins Associated With Laryngeal Carcinoma-Related Gene 1,THE LARYNGOSCOPE, Issue 2 2006Xiaopeng Zhang PhD Abstract Objectives: A novel gene, laryngeal carcinoma-related gene 1 (LCRG1), had the characteristics of tumor-suppressor genes. It was cloned in our laboratory. The objective was to find and characterize the proteins related to LCRG1 and to elucidate the molecular mechanism of LCRG1. Study Design: We used the established cell lines of Hep-2/LCRG1 (Hep-2 cells transfected by recombinant plasmid pcDNA3.1[+]/LCRG1) and Hep-2/pcDNA3.1(+) (Hep-2 cells transfected by control vector pcDNA3.1[+]) as cell models. Methods: Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization,quadruple time-of-flight MS/MS (ESI-Q-TOF MS/MS). Then the differential expression levels of partial identified proteins were determined by Western blotting analysis and quantitative real-time reverse transcriptase,polymerase chain reaction. Results: The results showed the attained 2-DE patterns of the two cell lines were well-resolved and reproducible. There were 1075 ± 43 and 1027 ± 23 protein spots observed in Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The average matching rate of the two cell lines was 91%. Twenty-six differentially expressed protein spots were identified (twenty spots for MALDI-TOF-MS, six spots for ESI-Q-TOF MS/MS). Most of the characterized proteins were characterized as the members of enzymes (phosphoglycerate mutase, manganese superoxide dismutase, and so on), transcription proteins (rho gdp dissociation inhibitor), and so on. Those identified proteins might contribute to the tumor-suppressive function of LCRG1. The differential expression levels of the partial proteins were confirmed by real-time polymerase chain reaction and Western blotting. Conclusions: We tentatively proposed those differentially expressed proteins were involved in the tumor-suppressive process related to LCRG1. These data will be helpful to elucidate the molecular mechanism of LCRG1. [source] Methylation-Associated Silencing of Death-Associated Protein Kinase Gene in Laryngeal Squamous Cell Cancer,THE LARYNGOSCOPE, Issue 8 2005Wei-Jia Kong MD Abstract Objectives/Hypothesis: Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It has been found that DAPK gene is frequently inactivated by its promoter hypermethylation in some cancers and tumor cell lines. However, it is not clear whether promoter hypermethylation of DAPK gene exists in laryngeal squamous cell cancer (LSCC). The aim of this study was to investigate the promoter methylation status of the DAPK gene in LSCC and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on Hep-2 cells, a human laryngeal cancer cell line, and on xenografts of Hep-2. Methods: Methylation-specific polymerase chain reaction (PCR) and reverse-transcription PCR techniques were used to determine the promoter methylation status and mRNA expression of DAPK gene in LSCC. Furthermore, Hep-2 cells in vitro and in vivo were treated by 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth. Results: Hypermethylation of DAPK gene promoter was found in 39 (67.2%) of 58 LSCC samples. There was no significant difference in the promoter hypermethylation rate among the samples of different histologic grades or samples from patients with different T stages. However, there was significant difference in methylation status of DAPK gene between the samples from patients in N0 stages and those from patients in N1 stages. No promoter hypermethylation of DAPK gene was found in any of the five normal laryngeal tissue samples. DAPK mRNA expression was not detected in tumor specimens with promoter hypermethylation. On the contrary, DAPK mRNA expression was observed in the unmethylated tumor specimens, specimens from tissues adjacent to the tumor, and normal laryngeal tissues samples. Promoter hypermethylation of DAPK gene was found, and no DAPK mRNA expression was detected in Hep-2 cells. DAPK mRNA expression in Hep-2 cells and xenografts could be restored by treating cells and xenografts with 5-Aza-CdR. The tumors' xenografts, induced by way of Hep-2 cell injection in nude mice treated with 5-Aza-CdR, were obviously smaller than those in nude mice treated with phosphate-buffered saline. Conclusions: Abnormal loss of DAPK expression could be associated with aberrant promoter region methylation in the LSCC. 5-Aza-CdR may slow the growth of Hep-2 cells in vitro and in vivo by reactivating tumor suppressor gene DAPK silenced by de novo methylation. [source] | |||||||||||||