Iap Gene Research Articles

P1-005: Tissue content of Suvivin mRNA and Survivin Expression in nonsmall cell lung cancer for diagnosis and prognosis.

Deregulation of apoptosis pathway is one of crucrial mechanisms causing tumorigenesis and driving cancer progression. Survivin is a member of the inhibitor of apoptosis, IAP gene family. It plays a dual role in both cell death suppression and cell cycle regulation at the G2/M phase. Its expression also contributes to resistance to chemo- and radiotherapy of the cancer cells. This study was aimed at determination of surviving mRNA expression semiquantitatively comparing to the surviving expression in tumor tissue of the nonsmall cell lung cancer. Correlation between survivin mRNA content and expression of the commercially available survivin in the tumor tissue and pathlogical parameters is also evaluated proving the significance of survivin in cancer diagnosis and prognosis. 22 cases of surgically resected nonsmall cell lung cancer were included during the year 2005 and the early 2006. Total RNA was extracted from fresh frozen tissues of both cancer and corresponding normal lung tissues. The formalin-fixed, paraffin-embedded cancer tissue has been tested with commercially available suvivin antibody immunohistochemcially. The correlation between mRNA expression and protein expression was evaluated with pathological parameters using Chi-squre test. Selective survivin expression in the lung cancer tissue not in the normal lung tissue makes it a potential diagnostic marker however, the prognostic marker to select the cases that might be chemoresponsive needs to verify with the other markers.

A Novel Multiplex PCR Assay for Rapid and Simultaneous Detection of Five Pathogenic Bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4 to 7 days to complete. Thus, this study was conducted to address the issue of time lag inherent in traditional methods by developing a novel PCR assay for each of five foodborne pathogenic bacteria. This new system consists of a simultaneous screening method using multiplex PCR in a single reaction tube for the rapid and sensitive detection of each of the five bacteria. Specific primers for multiplex PCR amplification of the Shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes were designed to allow simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella, respectively. To confirm the specificity of each primer pair for the respective target gene, three types of experiments were carried out using boiled cell lysates and their DNAs. In the multiplex PCR with mixed DNA samples, specific bands for corresponding genes were simultaneously detected from a single reaction. The detection of all five foodborne pathogenic bacteria could be completed in less than 24 h with this novel PCR method.

XIAP is highly expressed in esophageal cancer and its downregulation by RNAi sensitizes esophageal carcinoma cell lines to chemotherapeutics

X-linked inhibitor of apoptosis protein (XIAP) is the most potent member of the IAP gene family in terms of its ability to inhibit caspases and suppress apoptosis. In this study, we investigated the expression of XIAP in esophageal cancer tissues and cell lines, and found the elevated expression of XIAP in esophageal cancer tissues compared with normal tissues. Then we used small interfering RNA (siRNA) to block XIAP expression while evaluating the effect of XIAP siRNA on cell apoptosis, and the combined effects with Paclitaxel, Cisplatin, Fluorouracil, and Etoposide in XIAP high expression ESCC cell line KYSE150 and EC9706. The results showed that XIAP siRNA efficiently decreased XIAP expression and induced cell apoptosis. Treatment with XIAP siRNA in combination with Paclitaxel, Cisplatin, Fluorouracil, and Etoposide enhanced chemosensitivity. These results suggest that XIAP might be helpful for diagnosis of ESCC and XIAP siRNA combined with Paclitaxel, Cisplatin, Fluorouracil, and Etoposide may be a feasible strategy to enhance the effects of chemotherapy in patients with ESCC.

The genome sequence of the multinucleocapsid nucleopolyhedrovirus of the Chinese oak silkworm Antheraea pernyi

The complete genome sequence of the Liaoning isolate of the Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeMNPV) was determined. The viral genome size is 126,246 bp, relatively GC-rich (53.5% G + C), with 145 predicted open reading frames (ORFs) of more than 50 amino acids, accounting for more than 97% of the genome. 97% of the ORFs have predicted functions or homologues in other baculoviruses. There are six homologous repeat regions ( hrs) and two bro homologues, which are associated with regions of genome instability. The virus lacks the p35 homologue but encodes two members of the inhibitors of apoptosis ( iap) gene family. The presence of a gp64 homologue in the AnpeMNPV genome and results from gene parity plot and phylogenetic analysis using the 29 core baculovirus genes clearly classify AnpeMNPV as a group I NPV. The divergence of genome sequences in two geographical NPV isolates suggests of great genetic heterogeneity of baculovirus populations in nature.

Effect of hepatocyte growth factor (HGF) on the level of Survivin & XIAP expression in several human cancer cell lines, after treating with DNA damaging agent

Hepatocyte growth factor (HGF) has opposite biological activities in regulating apoptosis, also underlying molecular mechanisms are not clearly defined. We investigated HGF ability to inhibit cell death, which was induced by Doxorubicin, a DNA damaging agent. Also Survivin and XIAP mRNA levels were compared in HGF treated and non-treated cells. Cell proliferation and death were assessed using MTT assay and dye exclusion tests. Quantitative real-time PCR was used to evaluate Survivin and XIAP expression levels after treatment with HGF. ELISA was performed to quantify HGF secretion in the selected cancer cell lines media. HGF appeared to have inhibitory effect on Doxorubicin induced cell death in all of the studied cell lines. It had minimal effect on XAIP and Survivin expression levels in MRC-5, MOLT-4 and AGS cell lines; except for XIAP expression level in AGS cell line, which was increased substantially after treatment. Surprisingly, in KG-1 cell line, XIAP and Survivin expression levels were significantly reduced after HGF treatment. Although several members of IAP gene family are reported to play role in HGF mediated cytoprotective pathway, we showed that XIAP and Survivin do not seem to be involved.

Genotypic characterization of Listeria spp. isolated from fresh water fish

A total of 200 samples (muscles and viscera, 100 of each) of fresh water fish, walking catfish ( Clarias batrachus) were screened for Listeria spp. All the samples were subjected to a two-step enrichment followed by plating on selective media. Confirmation of the isolates was on the basis of biochemical characters, haemolysis on blood agar and Christie, Atkins, Munch Petersen test. A total of 39 isolates of Listeria spp. were recovered. Of these 26 (67%), 8 (21%), 3 (8%) and 2 (5%) were Listeria monocytogenes, Listeria seeligeri, Listeria grayi and Listeria welshimeri, respectively. The isolates were subjected to a PCR assay for detection of the virulence-associated genes individually or together. The plcA, actA, hlyA and iap genes were detected in six strains, three genes ( actA, hlyA and iap) in nine strains, the plcA, hlyA and iap in our strain, the hlyA and iap were in three strains, actA and hlyA in four strains, plcA and hlyA in our strain and hlyA in two strains. The hlyA and iap were also detected in L. seeligeri.

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh 7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

BackgroundThe absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure.ResultsIn this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells.ConclusionThe culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

Rabbit Meat as a Source of Bacterial Foodborne Pathogens

Even though worldwide production of rabbit meat is >1,000,000 tons, little information is available for rabbit meat microbiology. This study provides data on the prevalence of Salmonella, Escherichia coli O157:H7, Yersinia enterocolitica, Listeria spp., motile Aeromonas spp., and Staphylococcus aureus on rabbit meat. A total of 24 rabbit carcasses from two abattoirs and 27 rabbit meat packages from supermarket displays were examined. In addition to culturing methods, associated virulence genes were investigated by PCR in suspect isolates and samples. Neither Salmonella nor E. coli O157:H7 was detected. All samples were negative for virulence-associated invA, stx1, and stx2 genes. At one abattoir, two carcasses (3.9%) carried Y. enterocoliticayst−, and two were positive for the yst gene, although viable Y. enterocolitica cells were not recovered from these samples. Seven samples (13.7%) were contaminated with Listeria. Of them, three were positive for hly and iap genes (Listeria monocytogeneshly+/iap+), two carried Listeria seeligeri, one carried Listeria ivanovii, and one carried Listeria innocua. For detectable motile Aeromonas spp. (average count, 1.77±0.62 log CFU/g), the contamination rate was 35.3%, although ca. 90% of the samples were positive for the aerA and/or hlyA genes. The majority of aeromonad isolates were Aeromonas hydrophilaaerA+/hlyA+. Aeromonas caviae, Aeromonas popoffii, Aeromonas schubertii, and the two biovars of Aeromonas veronii were also isolated. The prevalence of S. aureus contamination (average count, 1.37±0.79 log CFU/g) was 52.9%. Among 27 S. aureus isolates, two harbored genes for staphylococcal enterotoxin B (seb), and two harbored genes for staphylococcal enterotoxin C (sec). The remaining isolates were negative for sea, seb, sec, sed, and see.

Evaluation of PCR-Based Identification and Genotyping Methods for the iap Genes in Listeria monocytogenes and other Listeria spp.

Evaluation of PCR-Based Identification and Genotyping Methods for the iap Genes in Listeria monocytogenes and other Listeria spp.

Multiplex PCR identification of Listeria monocytogenes isolates from milk and milk‐processing environments

AbstractA multiplex PCR procedure based on genes iap (coding for the invasion‐associated 60 kDa protein or p60) and hly (coding for listeriolysin O) of Listeria monocytogenes was to used to investigate the status of its contamination along the major milk‐processing environments. Duplex PCR amplified fragments of the iap gene at about 1.45 kb from all strains of major Listeria spp. tested and a 420 bp fragment of hly from L. monocytogenes reference strains. With triplex PCR, all L. monocytogenes strains exhibited a 420 bp fragment of hly as well as a 700 bp fragment of iap instead of its 1.45kb PCR product. The tentative L. monocytogenes isolates (n = 27) out of 566 samples of milk and milk‐processing environments in the PALCAM agar from cultures of buffered listeria enrichment broth were further subjected to both API and triplex PCR identification. Both methods identified the same 20 isolates as L. monocytogenes. The triplex PCR procedure detected as low as 3.2 × 101 cfu mL−1 of listerial cells even in the presence of L. innocua (108 cfu mL−1). It could detect 1.4 × 102 listerial cells mL−1 directly from milk artificially contaminated with the bacteria. Lower levels of L. monocytogenes cells in milk (1.45 × 101 to 1.45 × 100 cfu mL−1) could be detectable if the inoculated milk samples were cultured for 3–6 h. The bacterium was found not only in raw milk, sewage water and vessel surfaces but also in pasteurized milk. Copyright © 2005 Society of Chemical Industry

An RNA interference screen ide.jpgies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila

Innate immunity in vertebrates and invertebrates is of central importance as a biological programme for host defence against pathogenic challenges. To find novel components of the Drosophila immune deficiency (IMD) pathway in cultured haemocyte-like cells, we screened an RNA interference library for modifiers of a pathway-specific reporter. Selected modifiers were further characterized using an independent reporter assay and placed into the pathway in relation to known pathway components. Interestingly, the screen identified the Inhibitor of Apoptosis Protein 2 (IAP 2) as being required for IMD signalling. Whereas loss of DIAP 1, the other member of the IAP protein family in Drosophila, leads to apoptosis, we show that IAP 2 is dispensable for cell viability in haemocyte-like cells. Cell-based epistasis experiments show that IAP 2 acts at the level of Tak 1 (transforming growth factor-beta-activated kinase 1). Our results indicate that IAP gene family members may have acquired other functions, such as the regulation of the tumour necrosis factor-like IMD pathway during innate immune responses.

Discrimination of Listeria monocytogenes contaminated commercial Japanese meats

Discrimination was attempted on 14 Listeria monocytogenes strains isolated from commercially available Japanese pork and chicken. Examination of the isolates was performed by restriction fragment length polymorphism (RFLP) analysis of the chromosomal DNA and amplified products and comparison of the nucleotide sequences of the amplified products. A polymorphism region containing the repeated sequences in the iap gene was amplified by the polymerase chain reaction (PCR). The genetic analyses could discriminate the 14 isolates in combination with traditional serotyping, and some strains isolated from different meats were confirmed to have a genetically close relationship. Genetic analyses used in the present study would be useful for the elucidation of the pathogen tracks from contaminated sources to humans and of the ecological niche in the food environment.

A novel function of transcription factor α-Pal/NRF-1: Increasing neurite outgrowth

α-Pal/NRF-1 is a critical regulator of the promoter of human IAP/CD47 gene, a gene related to memory formation in rodents. However, its function in neurons was unknown. We found that stable or transient expression of full-length α-Pal/NRF-1 in human neuroblastoma IMR-32 cells significantly induced neurite outgrowth and increased the length of neurites both in medium containing 10% fetal bovine serum and in serum-free medium. In contrast, the dominant-negative mutant of α-Pal/NRF-1 inhibited the induction and extension of neurites. Ectopic expression of full-length α-Pal/NRF-1 also increased the induction of neurite outgrowth in primary mouse cortical neurons. The IAP antisense cDNA significantly inhibited the increase of neurite outgrowth by α-Pal/NRF-1. These findings indicate that a novel function of α-Pal/NRF-1 is to regulate neuronal differentiation, and that this function is mediated partly via its downstream IAP gene.

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.

PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid.

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.

Functional analysis of the inhibitor of apoptosis (iap) gene carried by the entomopoxvirus of Amsacta moorei.

The entomopoxvirus from Amsacta moorei (AmEPV) contains none of the commonly recognized vertebrate poxvirus apoptotic suppressor genes. However, AmEPV carries a single inhibitor of apoptosis (iap) gene (AMViap) not present in vertebrate poxviruses. The AMViap gene was active when coexpressed with the Drosophila proapoptotic gene hid in Ld652 cells and can rescue cells from apoptosis as shown by increased number of surviving cells and reduced levels of caspase-3-like activity. We also showed that expression of the AMViap gene rescued polyhedron production in Autographa californica M nucleopolyhedrovirus (AcMNPV)Deltap35-infected Sf9 cells during an otherwise abortive infection induced by apoptosis. Surprisingly, deletion of the AMViap gene from the AmEPV genome led to only a modest (10-fold) loss of virion production in infected Ld652 cells, indicating that the AMViap gene is nonessential for virus replication under these conditions. However, infection of Ld652 cells by AmEPV lacking a functional iap gene led to a more rapid induction of cytotoxicity and increased levels of caspase-3-like activity. Similar results were observed and were more pronounced in infected Sf9 and S2 cells. The purified AMVIAP protein also inhibits the enzymatic activities of human caspase-9 and caspase-3 in vitro. Our results indicate that while the AMViap gene was active in controlling apoptosis through the intrinsic pathway, the virus likely encodes additional proteins that also regulate apoptosis.

Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.

P21Waf1/Cip1 and the prevention of oxidative stress.

pulmonary dysfunction resulting from acute oxygen toxicity is due at least in part to the death of lung epithelial cells. Pulmonary epithelial cells are essential to the maintenance of the alveolar-capillary barrier, and compromise may lead to leakage of fluid and proteins in the airways and alveoli