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Michael J Gray

Publications and source records attributed to Michael J Gray.

At least 19 recordsLinked to original sources
PubMed2025

Genome-wide characterization of hypothiocyanite stress response in Escherichia coli.

UNLABELLED: Oxidative stress is one of the major methods of microbial population control and pathogen clearing by the mammalian immune system. The methods by which bacteria are able to escape damage by host-derived oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) have been relatively well described, while other oxidants' effects on bacteria and their genetic responses are not as well understood. Hypothiocyanite/hypothiocyanous acid (OSCN-/HOSCN) is one such oxidative stress agent. In this study, we used RNA-sequencing to characterize the global transcriptional response of Escherichia coli to treatment with HOSCN and the impact of deletions of the HOSCN resistance proteins RclA (HOSCN reductase), RclB, and RclC on that response. The HOSCN response of E. coli was different from the previously characterized responses of E. coli to other oxidants such as H2O2, superoxide, or HOCl and distinct from the reported responses of other bacteria such as Streptococcus pneumoniae and Pseudomonas aeruginosa to HOSCN. Strikingly, deletion of any one of the Rcl proteins had very similar effects on the transcriptional response to HOSCN, indicating that any disruption of HOSCN defense in E. coli results in similar impacts, despite the fact that we do not currently understand the mechanism(s) by which RclB and RclC contribute to that defense. IMPORTANCE: Understanding how bacteria sense and respond to oxidative stress provides insights into how our bodies interact with the microbial population within us. In this study, we have characterized the genetic response of E. coli to the important immune oxidant hypothiocyanite and investigated the role of rclABC genes in that response.

Escherichia coli↗
PubMed2006

Src activation of Stat3 is an independent requirement from NF-kappaB activation for constitutive IL-8 expression in human pancreatic adenocarcinoma cells.

Human pancreatic tumors often overexpress the angiogenesis-promoting factor Interleukin 8 (IL-8), in part due to overexpression of NF-kappaB, a frequent occurrence in pancreatic adenocarcinoma. In this study, we demonstrate that reducing c-Src kinase activity, through either pharmacologic inhibition or small interfering RNA-targeted reduction of Src expression, significantly decreased IL-8 expression (P < 0.05) without affecting NF-kappaB-mediated transcription, but by decreasing phosphorylation of STAT3. To ascertain whether Src-mediated expression of IL-8 was dependent on STAT3, we used stable clones expressing a dominant-negative isoform of STAT3 that inhibits endogenous STAT3 phosphorylation and subsequent DNA binding and STAT3-mediated gene expression or a constitutively activated isoform of STAT3. IL-8 expression was significantly lower in clones expressing the dominant-negative isoform and significantly increased in clones expressing the activated isoform (P < 0.05 for both). Pharmacologic inhibition of NF-kappaB activity significantly reduced basal IL-8 expression and tumor necrosis factor-induced IL-8 expression (P < 0.05 for both), yet NF-kappaB activity was not dependent on Src. We therefore suggest that Src activation, through phosphorylation of STAT3, and NF-kappaB are all required for expression of IL-8 a critical angiogenic-promoting factor in pancreatic adenocarcinomas.

Adenocarcinoma↗
PubMed2006

Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT. EXPERIMENTAL DESIGN: The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses. RESULTS: The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an approximately 8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and beta-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor kappaB. CONCLUSION: We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.

Antineoplastic Agents↗
PubMed2006

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells.

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Cell Line, Tumor↗
PubMed2006

Multidimensional liquid chromatography for sample characterisation.

While LC finds enormously widespread use in almost all areas of chemical science, the technique is limited as a means of identification because compounds do not elute with unique retention times. This limitation spurred the growth of hyphenated instrumental methods of analysis, such as LC-MS/MS, which because of the MS/ MS detection became a method of identification. However, techniques like LC-MS/ MS are specialised and require high initial purchase and running costs, inhibiting the more widespread growth of the technique. In an attempt to increase the separation power of LC, multi-dimensional LC was developed. This expanded the separation space and subsequently has allowed the development of methods with fingerprinting ability due to the lower probability of component overlap. The work in this study illustrates the application of 2-D LC as a means of chemical fingerprinting. We employed a sample base of various low molecular weight oligostyrenes and their diastereomers that represent a population of compounds whose selectivities in a one-dimensional separation are almost unity and hence essentially impossible to separate. Yet in a 2-D domain almost all individual components occupy unique 2-D retention times.

Journal Article↗
PubMed2006

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades.

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

Base Sequence↗
PubMed2006

The role of nitric oxide in mediating tumour blood flow.

Nitric oxide (NO) is a ubiquitous molecule with a myriad of physiological and pathophysiological roles. It has numerous direct and indirect effects on tumour vasculature as both a regulatory and effector molecule. NO affects tumour blood flow through its effects on tumour angiogenesis, vascular tone and vascular permeability, partly via its interaction with vascular endothelial growth factor. In this review, the authors examine the basic tenants of NO biology, the association of NO with tumour progression, and the role NO plays in mediating alterations in vascular functions in tumours.

Animals↗
PubMed2006

Attributing risk to Listeria monocytogenes subgroups: dose response in relation to genetic lineages.

The objective of this study was to evaluate the hypothesis that the dose-response relationship for Listeria monocytogenes in humans varies with genotypic lineage or subtype. The linkages between molecular subtyping data and enumeration data for L. monocytogenes subtypes in foods consumed by the at-risk population were examined to test this hypothesis. We applied a conditional probability model to conduct a subtype-specific dose-response analysis, with the focus on invasive listeriosis. L. monocytogenes differed not only in the molecular subtype and lineage but also in the contamination level when isolates of the pathogen occurred in retail samples of ready-to-eat foods. Using the exponential model parameter r-value as a measure (essentially the probability of a single cell causing illness), we found that the virulence varied among L. monocytogenes lineages by several orders of magnitude. Under the assumptions made, for L. monocytogenes lineages I and II the consumption of a single cell would result in listeriosis with log average probabilities of -7.88 (equivalent to once in 10(7.78) times) and -10.3, respectively, as compared with -9.72 for L. monocytogenes independent of subtype. A greater difference in r-values was found for selected ribotypes. The uncertainty about the r-value estimates was small compared with the large differences in the virulence parameters themselves. Thus, for L. monocytogenes both subtype and the number of cells consumed matter, highlighting the usefulness of considering both exposure concentration and subtype prevalence in dose-response analysis. As advances are made in molecular subtyping and quantitative tools for dose-response analysis, further studies integrating genomic data into quantitative risk assessments will enable better attribution of disease risk to L. monocytogenes subtypes.

Animals↗
PubMed2005

Study of the selectivity of reversed-phase columns for the separation of polycarboxylic acids and polyphenol compounds.

A systematic investigation was undertaken into the relative separation performance of five reversed-phase chromatography columns including some commercially new hybrid packed columns for a series of polycarboxylic acids and polyphenol compounds. Information theory (IT) and factor analysis (FA), together with a basic evaluation of retention information (band shape, retention factor and elution order) were used to compare four columns to a conventional C18 column. The results revealed very little difference in retention behaviour between the Phenomenex Aqua C18 column, the Waters XTerra RP C18 column, and the conventional Phenomenex Luna C18 column. However, there were notable differences in the retention processes between the Phenomenex Synergi polar-RP column, which is an ether-linked phenyl base with polar endcapping, and the Luna C18 column. The most significant differences were observed between the Luna C18 column and a Phenomenex Luna Cyano column. However, the limited degree of retention of the polycarboxylic acids and polyphenol compounds on the Luna Cyano column permits only limited use for the separation of these types of compounds. Overall, the Phenomenex Synergi polar-RP column exhibited the best performance for the separation of the test solutes compared to that of the conventional C18 column, with IT yielding an Informational Similarity of 0.99 and FA a moderate correlation coefficient of 0.70. The Phenomenex Synergi polar-RP column gave the best peak shape and offered substantial selectivity differences thereby providing a good alternative over the conventional C18 column for separating polycarboxylic acids and polyphenols.

Adsorption↗
PubMed2005

Expression and activity of SRC regulate interleukin-8 expression in pancreatic adenocarcinoma cells: implications for angiogenesis.

Interleukin-8 (IL-8) is an angiogenic factor that promotes growth of pancreatic tumors. The purpose of this study was to determine if c-Src, a protein tyrosine kinase frequently overexpressed in pancreatic cancer, regulated IL-8 expression and to elucidate the Src-mediated signaling pathways that contribute to angiogenesis in pancreatic adenocarcinoma cells. In a panel of pancreatic cancer cell lines, expression of total and activated Src correlated with IL-8 production. Furthermore, ectopic expression of activated Src in PANC-1 cells with low endogenous Src activity significantly increased IL-8 production (P < 0.005). In contrast, pharmacologic inhibition of endogenous c-Src kinase activity or small interfering RNA-mediated "knockdown" of c-Src expression in L3.6pl cells with high Src expression and activity caused significant decreases in IL-8 production (P < 0.005). Inhibition of c-Src activity resulted in decreased phosphorylation of Akt, p38, and extracellular signal-regulated kinase (Erk)-1/2. Significant (P < 0.005) dose-dependent decreases were observed in IL-8 expression by inhibiting Src-dependent signaling molecules Erk-1/2 and p38 but not phosphatidylinositol 3-kinase. To assess the relevance of Src inhibition to angiogenesis, in vivo gelfoam assays were done. Robust infiltration of vessels was observed in gelfoam saturated with conditioned medium from pancreatic carcinoma cells. This angiogenesis was nearly abrogated in gelfoams saturated with conditioned medium from cells treated with the Src family kinase inhibitor, PP2 (P < 0.001). Thus, c-Src regulates critical "downstream" signaling pathways that contribute to expression of IL-8 in human pancreatic tumor cells, suggesting c-Src may be a target for therapeutic intervention in pancreatic adenocarcinoma.

Adenocarcinoma↗
PubMed2005

Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines.

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1) is one of three receptor tyrosine kinases for VEGF, a key regulator of angiogenesis in cancer. Although VEGFRs initially were believed to be expressed exclusively on endothelial cells (ECs), recent studies have demonstrated the presence of VEGFR-1 on non-EC types. The authors hypothesized that VEGFR-1 is present and functional in pancreatic carcinoma cells, contributing to the malignant phenotype. METHODS: The authors assessed the expression of VEGFR-1 and its ligands in 11 pancreatic carcinoma cell lines by reverse-transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blot analysis. The function of VEGFR-1 was evaluated by treating two representative cell lines with VEGF-B, a selective ligand for VEGFR-1, and/or a specific anti-VEGFR-1 antibody and assessing the effects on signaling, migration, invasion, and proliferation. RESULTS: All 11 pancreatic carcinoma cell lines expressed VEGFR-1 mRNA and protein, as well as the VEGFR-1 ligands VEGF-A and VEGF-B. Two representative cell lines (L3.6 and Panc-1) exhibited VEGF-B-induced mitogen-activated protein kinase signaling. A VEGFR-1 neutralizing antibody abrogated signaling, confirming that the ligand effect was mediated through VEGFR-1. VEGFR-1 stimulation by VEGF-A or VEGF-B was found to promote migration in both cell lines. Panc-1 cells also demonstrated enhanced Matrigel invasion after VEGFR-1 stimulation. VEGFR-1-dependent migration and invasion were blocked by the VEGFR-1 neutralizing antibody. VEGFR-1 activation did not appear to enhance cell proliferation. CONCLUSIONS: VEGFR-1 appears to be expressed ubiquitously in pancreatic carcinoma cell lines, in which it induces signaling and promotes migration and invasion. Overexpression of VEGF in tumors may activate tumor cells bearing VEGFR-1 via an autocrine pathway. Agents targeting VEGF or its receptors may have a dual inhibitory effect on tumor growth by suppressing both angiogenesis and tumor cell function.

Blotting, Western↗
PubMed2005

Induction of arginase I transcription by IL-4 requires a composite DNA response element for STAT6 and C/EBPbeta.

Arginine metabolism in macrophages during infection and inflammation is complex, owing to differential regulation of inducible nitric oxide synthase (iNOS) and arginases by cytokines and other agents. Changes in levels of Th2 cytokines such as interleukin-4 (IL-4) can play important roles in these conditions via effects on arginine metabolism. IL-4 alters macrophage arginine metabolism by inducing arginase I expression and inhibiting nitric oxide production. To determine the molecular basis for induction of arginase I, the promoter of the murine arginase I gene was cloned and analyzed by transfection in RAW 264.7 macrophage cells. IL-4 induction required a composite response element containing STAT6 and C/EBP sites located 2.86 kb upstream of the transcription start site. Competition experiments showed that STAT6 and C/EBPbeta bind to the STAT6 and C/EBP sites non-cooperatively. Elucidation of the mechanisms involved in regulation of arginase I transcription may provide a basis for developing strategies to modulate arginase expression in Th2 cytokine-predominant diseases.

Animals↗
PubMed2005

Molecular differences between deuterated and protonated polystyrenes using reversed-phase high-performance liquid chromatography.

Isotopic substitution is a technique used to highlight particular bonds within a molecule for kinetic, spectroscopic and structure analysis. It is presumed that although some properties such as stretching frequencies will not be the same for substituted analogues, the chemical interactions will not vary appreciably as a function of labelling. Reversed-phase liquid chromatography has been used to demonstrate that there are significant differences between the chromatographic behaviour of a sequence of deuterated and protonated oligomeric polystyrenes. Two-dimensional reversed-phase liquid chromatography was used to show that even the diasteromers of the oligomers (n = 5) have retention mechanisms that are dependent on the subtle changes to the molecular conformation and electronic structure, which are a consequence of deuteration.

Chromatography, High Pressure Liquid↗
PubMed2005

Utilising retention correlation for the separation of oligostyrenes by coupled-column liquid chromatography.

The separation of n = 2-5 n-butyloligostyrenes has been illustrated by reversed-phase reversed-phase (RP-RP) coupled-column liquid chromatography. The coupled-column separation has been achieved by use of a C18 column with methanol as the mobile phase followed by a DiamondBond C18 column with acetonitrile (ACN) mobile phase. The DiamondBond C18 is a hybrid carbon clad zirconia (CCZ)-C18 stationary phase. Unlike a C18-carbon clad zirconia two-dimensional liquid chromatographic system, which is orthogonal, the C18 and DiamondBond C18 columns combination exhibit correlations based upon the molecular weight of n-butyloligostyrenes. Using an alternative strategy to two-dimensional liquid chromatography, the molecular weight dependence displayed by both the C18 column and DiamondBond C18 has been used to increase throughput or decrease analysis time in the analysis of the n-butyloligostyrenes. However, this is at the expense of a portion of the two-dimensional peak capacity displayed by the C18-carbon clad zirconia system.

Chromatography, Liquid↗
PubMed2005

Neuropilin-1 suppresses tumorigenic properties in a human pancreatic adenocarcinoma cell line lacking neuropilin-1 coreceptors.

Neuropilin-1 (NRP-1) was first described as a coreceptor implicated in neuronal guidance that bound members of the semaphorin/collapsin family. NRP-1 is also expressed in endothelial cells and is believed to promote angiogenesis by acting as a coreceptor with vascular endothelial growth factor (VEGF) receptor 2. Recent studies suggest that NRP-1 can function through both a VEGF-dependent and VEGF-independent fashion. Expression of NRP-1 has been shown in many human tumors, including pancreatic adenocarcinomas. The exact role of NRP-1 in tumor cells is unknown, particularly in cells that lack the NRP-1 coreceptors VEGF receptor 2 and Plexin-A1. To discern the regulatory role(s) of NRP-1 in pancreatic adenocarcinoma that lack these coreceptors, we overexpressed both full-length NRP-1 and a deletion form of NRP-1 that does not interact with semaphorin or VEGF. Overexpression of either isoform reduced several key tumorigenic properties, including anchorage-independent cell growth and migration in vitro, and resulted in reduced tumor incidence and tumor volume in vivo. Conversely, reduction of NRP-1 expression by small interfering RNA targeting led to enhanced tumor growth. Thus, NRP-1 may play distinct growth regulatory roles in different tumor types, and altering NRP-1 expression or function may be a means of influencing the growth of pancreatic cancers.

Adenocarcinoma↗
PubMed2005

HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas.

Hypoxia stimulates a number of pathways critical to cancer cell survival, including the activation of vascular endothelial growth factor (VEGF) transcription. In normal fibroblasts, hypoxia-induced activation of the protein tyrosine kinase, Src, is required for VEGF expression. We show here in both pancreatic and prostate carcinoma cell lines cobalt chloride (used to mimic hypoxia) -induced VEGF expression requires Src activation and leads to increased steady-state levels of HIF-1alpha and increased phosphorylation of signal and transducer of transcription 3 (STAT3). STAT3 and hypoxia-inducible factor (HIF)-1alpha bind simultaneously to the VEGF promoter, where they form a molecular complex with the transcription coactivators CBP/p300 and Ref-1/APE. Expression of activated Src from an inducible promoter is sufficient to increase VEGF expression and form these STAT3/HIF-1alpha-containing promoter complexes. Inhibition of DNA binding by expression of either STAT3 or HIF-1alpha dominant negative mutants significantly reduces VEGF expression. These data suggest that the binding of both STAT3 and HIF-1alpha to the VEGF promoter is required for maximum transcription of VEGF mRNA following hypoxia.

Autoradiography↗