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PubMed2005

Farnesyltransferase inhibitor SCH-66336 downregulates secretion of matrix proteinases and inhibits carcinoma cell migration.

The ras oncogenes are among those most frequently found in human cancers. Blocking Ras farnesylation is a promising strategy for arresting cancer growth. Ras activates several signaling pathways with key roles in cellular proliferation, invasion, metastasis and angiogenesis. Furthermore, proteolytic activities of matrix proteinases such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) are regulated by Ras isoforms. Thus, we investigated the effects of SCH-66336, a farnesyltransferase inhibitor, on secretion of components of the plasminogen activation system as well as on the gelatinases MMP-2 and MMP-9, which play pivotal roles in matrix remodeling. SCH-66336 up to 5 microM did not significantly alter the viability of prostate (PC-3) and renal (Caki-1) cancer cells incubated in serum-depleted medium. SCH-66336 partly inhibited the processing of H-Ras, while levels of mature N-Ras and K-Ras remained unaffected. Under these noncytotoxic conditions, uPA and tPA levels were lowered in culture medium but raised in cell lysates, suggesting inhibition of trafficking pathways. In contrast, SCH-66336 had no effect on uPAR expression or on secreted PAI-1 levels. As expected, the reduction of uPA and tPA activities by SCH-66336 inhibited the conversion of plasminogen to plasmin by about 25% in PC-3 cells. SCH-66336 also inhibited the levels of secreted pro-MMP-2 and pro-MMP-9 as well as the release of their inhibitors TIMP-1 and TIMP-2. SCH-66336 decreased both the adhesion and even more so the migration of PC-3 cells on gelatin. Thus, SCH-66336 inhibited farnesylation in both cancer cell types, and H-Ras functions should be reduced by the drug. In addition, the lower levels of secreted proteinases in the presence of SCH-66336 suggest that reduced matrix remodeling and cell migration should occur in treated tumors.

Alkyl and Aryl Transferases↗
PubMed2004

Multistep and multifactorial carcinogenesis: when does a contributing factor become a carcinogen?

Our greatest successes in fighting cancer derive from the identification and removal or inactivation of carcinogenic substances, and from the identification and removal of pre-malignant lesions. In comparison, our successes at treating already formed malignancies have been minimal. Therefore, emphasis should be put in identifying and removing pre-malignant lesions, and in the identification and removal of those agents that cause or contribute to cancer development. It is important to target initiators, co-carcinogens and promoters, since by removing any one of them, tumor growth may be prevented. Identification of these agents is difficult. Epidemiological studies largely study cancer after it has occurred. It would be preferable to identify potential carcinogenic substances at an earlier stage before they have caused a large number of malignancies and thus become identifiable by epidemiology. During the past three decades, we have accumulated an impressive amount of evidence concerning molecular pathways that when altered contribute to malignant growth. It is time that we start applying this knowledge to the identification of human carcinogens. Here, we review the molecular changes that are required for carcinogenesis and propose some criteria that, in the absence of epidemiological evidence, can be used to identify agents that cause or contribute to human cancer development. In the absence of epidemiological evidence, a given agent should be considered a human carcinogen when: (1) the agent causes or contributes to the development of tumors in animals that are of the same type as those tumors associated with exposure to the agent in humans; (2) the agent transforms or contributes to the transformation of human cells in culture and these cells are of the same type from which associated human malignancies arise; (3) there is molecular evidence that the agent interferes with one or more key molecular pathways in human cells which leads to the formation of human cancer.

Animals↗
PubMed2003

Amifostine induces anaerobic metabolism and hypoxia-inducible factor 1 alpha.

PURPOSE: The cytoprotective mechanism of amifostine (WR-2721) implies free radical scavenging and DNA repair activities. We investigated additional cytoprotective pathways involving intracellular hypoxia and the activation of the hypoxia-inducible factor (HIF) pathway, a key transcription factor regulating glycolysis, angiogenesis and apoptosis, which is also linked with radioresistance. MATERIALS AND METHODS: The glucose and oxygen levels in the peripheral blood of patients receiving 1000 mg amifostine were determined at various time-points in order to investigate the metabolic changes induced by amifostine. MDA468 breast tumor cell lines were incubated with a high amifostine concentration (10 m M) to overcome the natural resistance of cancer cells to influx of the non-hydrolyzed WR-2721, and the HIF1 alpha protein levels were determined by Western blot analysis. In vivo experiments with Wistar rats were performed in order to assess immunohistochemically changes in the intracellular accumulation of HIF1 alpha induced by amifostine (200 mg/kg). RESULTS: By 30 min following amifostine administration, the hemoglobin oxygen saturation and pO(2) levels had increased in the peripheral blood while glucose levels had reduced, providing evidence that normal tissue metabolism switches to glycolytic pathways. Incubation of cell lines with amifostine resulted in HIF1 alpha induction. In Wistar rats administration of amifostine resulted in increased HIF1 alpha accumulation in normal tissues. CONCLUSIONS: Since it is doubtful whether dephosphorylation of amifostine to the active metabolite WR-1065 occurs within tumoral tissues (an acidic environment that lacks vascular alkaline phosphatase activity), intracellular hypoxia and upregulation of HIF1 alpha represents an additional, normal tissue-specific, amifostine cytoprotective pathway.

Amifostine↗
PubMed2006

Protection by EGb 761 against beta-amyloid-induced neurotoxicity: involvement of NF-kappaB, SIRT1, and MAPKs pathways and inhibition of amyloid fibril formation.

Abeta peptide-induced toxicity is mediated through oxidative stress and is associated with an activation of intracellular signaling such as the redox-sensitive transcription factor NF-kappaB and MAPK pathways. We demonstrate on neuroblastoma cell line N2a that EGb 761 could prevent the activation of NF-kappaB, ERK1/2, and JNK pathways induced by Abeta. Furthermore, our results show that EGb 761 can also activate SIRT1. This activation could explain the reduction of NF-kB activity by promoting the deacetylation of Lys310 of subunit p65. On the other hand, aggregation of Abeta to insoluble fibrils is a crucial step in Abeta-induced neurotoxicity. Using fluorescence spectroscopy with thioflavin T and electron microscopy, we demonstrate that EGb 761 and its flavonoid fraction (CP 205) could prevent the Abeta fibril (fAbeta) formation in vitro. Finally we show that Abeta is less toxic to N2a neuroblastoma cells when the peptide is previously incubated with the flavonoid fraction or EGb 761 during the fibril formation period. On the other hand, the ginkgolide compound BN 52021 was not able to prevent fAbeta formation. Interestingly it could also protect cells against Abeta toxicity. Our study demonstrates that the protection of neuronal cells by EGb 761 against Abeta could involve different mechanisms as the regulation of several key intracellular pathways and the inhibition of fAbeta formation and implicate more than its free radical scavenging property.

Amyloid beta-Peptides↗
PubMed2003

Gene expression signatures identify novel regulatory pathways during murine lung development: implications for lung tumorigenesis.

Oligonucleotide array based analysis was conducted to examine the temporal pattern of gene expression across the various stages of lung development to identify regulatory pathways at key developmental time points. Whole embryo total RNA or embryonic lung total RNA was harvested from A/J mice at seven developmental stages. To investigate changes in gene expression during lung development, four samples from each stage were examined using Affymetrix U74Av2 murine oligonucleotide microarrays. From the over 12,000 genes and ESTs represented on the array, 1346 genes and ESTs were identified as having a significant change in expression between at least one time point and the others (p<0.001, Kruskal-Wallis test). Within this group of approximately 1300 genes, four patterns of expression were seen: (1) upregulation during the embryonic period of development (up-down); (2) upregulation during the postnatal period of lung development (down-up) and (3) fluctuating expression, up initially, down for one or more time points, and then up again (up-down-up); and (4) vice versa (down-up-down). Expression patterns of genes previously reported to be involved in pulmonary development were also examined. Using the pathway visualisation tool, GenMapp, at least three regulatory pathways were found to contain clusters of differentially expressed genes: Wnt signalling, cell cycle, and apoptosis. Furthermore, we have shown that many of the genes involved in lung development are either known oncogenes or tumour suppressor genes altered in lung cancer, such as Cyr61, Rassf1a, and Dutt1/Robo1, or putative lung cancer genes. In addition, the genes identified pertinent to early development may also serve as candidate susceptibility genes for various inherited lung cancer disorders as well as for various heritable disorders of lung development. These results will contribute to our understanding of novel aspects of the regulatory machinery for embryonic lung development and of the genes involved in lung tumorigenesis.

Animals↗
PubMed2005

Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response.

Cyclin-dependent kinases (Cdk) promote cell proliferation, are often deregulated in human cancers, and are targets of ongoing cancer chemotherapy trials. We show here that Cdk activity is also required in human cells to maintain function of the Chk1 pathway, a key component of the response to DNA damage or stalled replication. Chk1 expression was markedly reduced in primary fibroblasts and U2OS osteogenic sarcoma cells by treatment with small molecule Cdk inhibitors or induction of a dominant-negative mutant of Cdk2. The findings of decreased Chk1 activity and accumulation of Cdc25A, a protein targeted for degradation by Chk1, confirmed that Chk1 function was impaired. Furthermore, Cdk inhibition triggered a DNA damage response, characterized by the accumulation of activated forms of ATM and Chk2 as well as nuclear foci containing phosphorylated substrates of ATM/ATR, including histone H2AX (gammaH2AX). Time course experiments showed that the bulk of ATM activation followed Chk1 down-regulation. Chk1 RNA interference combined with partial inhibition of DNA replication was sufficient to evoke the DNA damage response. Conversely, ectopic expression of Chk1 blunted induction of gammaH2AX foci by Cdk inhibitors, indicating that Chk1 down-regulation was necessary to elicit the full phenotype. Finally, both Cdk and Chk1 inhibitors enhanced the cytotoxity of etoposide, a DNA-damaging agent. These results define a pathway through which Cdk inhibition can mediate DNA damage and potentially enhance the efficacy of extant cancer chemotherapies.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

Cardiotrophin-1 defends the liver against ischemia-reperfusion injury and mediates the protective effect of ischemic preconditioning.

Ischemia-reperfusion (I/R) liver injury occurs when blood flow is restored after prolonged ischemia. A short interruption of blood flow (ischemic preconditioning [IP]) induces tolerance to subsequent prolonged ischemia through ill-defined mechanisms. Cardiotrophin (CT)-1, a cytokine of the interleukin-6 family, exerts hepatoprotective effects and activates key survival pathways like JAK/STAT3. Here we show that administration of CT-1 to rats or mice protects against I/R liver injury and that CT-1-deficient mice are exceedingly sensitive to this type of damage. IP markedly reduced transaminase levels and abrogated caspase-3 and c-Jun-NH2-terminal kinase activation after I/R in normal mice but not in CT-1-null mice. Moreover, the protective effect afforded by IP was reduced by previous administration of neutralizing anti-CT-1 antibody. Prominent STAT3 phosphorylation in liver tissue was observed after IP plus I/R in normal mice but not in CT-1-null mice. Oxidative stress, a process involved in IP-induced hepatoprotection, was found to stimulate CT-1 release from isolated hepatocytes. Interestingly, brief ischemia followed by short reperfusion caused mild serum transaminase elevation and strong STAT3 activation in normal and IL-6-deficient mice, but failed to activate STAT3 and provoked marked hypertransaminasemia in CT-1-null animals. In conclusion, CT-1 is an essential endogenous defense of the liver against I/R and is a key mediator of the protective effect induced by IP.

Alanine Transaminase↗
PubMedDate not supplied

Clinical proteomics: from biomarker discovery and cell signaling profiles to individualized personal therapy.

The discovery of new highly sensitive and specific biomarkers for early disease detection and risk stratification coupled with the development of personalized "designer" therapies holds the key to future treatment of complex diseases such as cancer. Mounting evidence confirms that the low molecular weight (LMW) range of the circulatory proteome contains a rich source of information that may be able to detect early stage disease and stratify risk. Current mass spectrometry (MS) platforms can generate a rapid and high resolution portrait of the LMW proteome. Emerging novel nanotechnology strategies to amplify and harvest these LMW biomarkers in vivo or ex vivo will greatly enhance our ability to discover and characterize molecules for early disease detection, subclassification and prognostic capability of current proteomics modalities. Ultimately genetic mutations giving rise to disease are played out and manifested on a protein level, involving derangements in protein function and information flow within diseased cells and the interconnected tissue microenvironment. Newly developed highly sensitive, specific and linearly dynamic reverse phase protein microarray systems are now able to generate circuit maps of information flow through phosphoprotein networks of pure populations of microdissected tumor cells obtained from patient biopsies. We postulate that this type of enabling technology will provide the foundation for the development of individualized combinatorial therapies of molecular inhibitors to target tumor-specific deranged pathways regulating key biologic processes including proliferation, differentiation, apoptosis, immunity and metastasis. Hence future therapies will be tailored to the specific deranged molecular circuitry of an individual patient's disease. The successful transition of these groundbreaking proteomic technologies from research tools to integrated clinical diagnostic platforms will require ongoing continued development, and optimization with rigorous standardization development and quality control procedures.

Biomarkers↗
PubMed2001

myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol.

myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzyme's activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.

Amino Acid Sequence↗
PubMed2004

Peroxisome proliferator-activated receptor gamma ligands inhibit Rho/Rho kinase pathway by inducing protein tyrosine phosphatase SHP-2.

Although peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells (RASMC), Rho kinase stimulated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone, and these effects were explained by the inhibition of the Rho translocation to the cell membrane. We evaluated the role of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. Furthermore, 4-week treatment with pioglitazone lowered systolic blood pressure in spontaneously hypertensive rats (SHR) and suppressed the Rho/Rho kinase activity in aortic tissues isolated from SHR. Consistently, the expression of SHP-2 was upregulated in vascular tissues from pioglitazone-treated SHR. The phosphorylated Vav was increased in SHR, compared with that in normotensive Wistar-Kyoto rats (WKY), which was mitigated by pioglitazone. Finally, both basal and angiotensin II-stimulated levels of Rho kinase activity were greater in RASMC from SHR than those from WKY, and the enhanced Rho kinase activity was blocked by pioglitazone or troglitazone in both strains. Collectively, PPARgamma ligands inhibit the Rho/Rho kinase pathway through upregulation of cytosolic SHP-2 expression and inactivation of Vav, and may contribute to the hemodynamic, in addition to metabolic, action in hypertensive metabolic syndrome. The full text of this article is available online at http://circres.ahajournals.org.

Angiotensin II↗
PubMed2002

Expression and regulation of WNT5A and WNT5B in human cancer: up-regulation of WNT5A by TNFalpha in MKN45 cells and up-regulation of WNT5B by beta-estradiol in MCF-7 cells.

WNT signaling pathway plays key roles in carcinogenesis and embryogenesis, and WNT signaling molecules are potent targets for diagnosis, prevention and treatment of cancer as well as for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14 and WNT14B/WNT15 using bioinformatics and cDNA-PCR. We have also reported frequent up-regulation of WNT2 and WNT5A in primary gastric cancer, which is probably due to cancer-stromal interaction. Here, expression and regulation of WNT5A and WNT5B in human cancer were investigated. WNT5A was relatively highly expressed in TE6 and TE10 among 12 esophageal cancer cell lines, and WNT5B was expressed in the majority of esophageal cancer cell lines. Among 7 pancreatic cancer cell lines, WNT5A was up-regulated in Hs700T, and WNT5B in PANC-1. WNT5A, but not WNT5B, was up-regulated by TNFalpha in MKN45 cells derived from gastric cancer. WNT5B, but not WNT5A, was up-regulated by beta-estradiol in MCF-7 cells derived from breast cancer. WNT5A and WNT5B were expressed together in 5 embryonal tumor cell lines, and were slightly down-regulated by all-trans retinoic acid in NT2 cells. Up-regulation of WNT5A and WNT5B in several types of human cancer expressing FZD5 might lead to more malignant phenotype through activation of the beta-catenin - TCF pathway.

Animals↗
PubMed2001

Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemical and genetic characterization of key enzymes.

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P. horikoshii genome. This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli. The recombinant product of gene PH0927 catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute alpha-mannosylglycerate. The MPG synthase and the MPG phosphatase were specific for these substrates. Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphate-guanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to alpha-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100 degrees C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100 degrees C and between pH 5.2 and 6.4. This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon.

Amino Acid Sequence↗
PubMed2005

Femtomolar transition state analogue inhibitors of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli.

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.

Deoxyadenosines↗
PubMed2005

Inflammatory pathway analysis using a high content screening platform.

High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response.

Anthracenes↗
PubMed2005

The extracellular zinc-sensing receptor mediates intercellular communication by inducing ATP release.

Taste and salivary secretion disorders have been linked to zinc deficiency, indeed zinc is found in secretory granules in the salivary gland. The signaling role for the zinc release in this tissue, however, is poorly understood. Here, we address the signaling pathways and physiological role of the zinc-sensing receptor, ZnR, in the ductal salivary gland cell line, HSY. Exposure of these cells to zinc triggered intracellular Ca2+ release from thapsigargin-sensitive stores. The G alpha q inhibitor, YM-254890 (1 microM), eliminated the Zn2+-dependent Ca2+ response, demonstrating that ZnR is a G alpha q-coupled receptor. Dose-response curves yielded an apparent K0.5 of 36 microM and a Hill coefficient of 7 in the absence of extracellular Ca2+, and K0.5 of 55 microM with a Hill coefficient of 3 in its presence. This indicates that although Zn2+ is essential for ZnR activation, Ca2+ may affect the receptor co-operativity. The homologous desensitization pattern of ZnR was characterized by pre-exposure of cells to Zn2+ at concentrations found to activate the receptor. Re-exposure of cells to Zn2+ elicited an attenuated Zn2+-dependent Ca2+ response for at least 3 h, indicating that the ZnR is strongly desensitized by Zn2+. Finally, we studied the paracrine affects of ZnR using a co-culture consisting of the HSY cells and vascular smooth muscle cells (VSMCs). While no Zn2+-dependent Ca2+ release was observed in VSMC alone, application of Zn2+ to the co-culture induced a Ca2+ rise in both HSY cells and VSMC. This Ca2+ rise was inhibited by the ATP scavenger, apyrase. Taken together, our results demonstrate that ZnR activity is monitored in salivary cells and is modulated by extracellular Ca2+. We further show that ZnR enhances secretion of ATP, thereby linking zinc to key signaling pathways involved in modification of salivary secretions by the ductal cells.

Adenosine Triphosphate↗
PubMed2003

Preferential metabolic activation of N-nitrosopiperidine as compared to its structural homologue N-nitrosopyrrolidine by rat nasal mucosal microsomes.

N-Nitrosopiperidine (NPIP) is a potent rat nasal carcinogen whereas N-nitrosopyrrolidine (NPYR), a hepatic carcinogen, is weakly carcinogenic in the nose. NPIP and NPYR may be causative agents in human cancer. P450-catalyzed alpha-hydroxylation is the key activation pathway by which these nitrosamines elicit their carcinogenic effects. We hypothesize that the differences in NPIP and NPYR metabolic activation in the nasal cavity contribute to their differing carcinogenic activities. In this study, the kinetics of tritium-labeled NPIP or NPYR alpha-hydroxylation mediated by Sprague-Dawley rat nasal olfactory or respiratory microsomes were investigated. To compare alpha-hydroxylation rates of the two nitrosamines, tritiated 2-hydroxytetrahydro-2H-pyran and 2-hydroxy-5-methyltetrahydrofuran, the major NPIP alpha-hydroxylation products, and tritiated 2-hydroxytetrahydrofuran, the major NPYR alpha-hydroxylation product, were quantitated by HPLC with UV absorbance and radioflow detection. These microsomes catalyzed the alpha-hydroxylation of NPIP more efficiently than that of NPYR. K(M) values for NPIP were lower as compared to those for NPYR (13.9-34.7 vs 484-7660 muM). Furthermore, catalytic efficiencies (V(max)/K(M)) of NPIP were 20-37-fold higher than those of NPYR. Previous studies showed that P450 2A3, present in the rat nose, also exhibited this difference in catalytic efficiency. For both types of nasal microsomes, coumarin (100 muM), a P450 2A inhibitor, inhibited NPIP and NPYR alpha-hydroxylation from 63.8 to 98.5%. Furthermore, antibodies toward P450 2A6 inhibited nitrosamine alpha-hydroxylation in these microsomes from 68.8 to 78.4% whereas antibodies toward P450 2E1 did not inhibit these reactions. Further immunoinhibition studies suggest some role for P450 2G1 in NPIP metabolism by olfactory microsomes. In conclusion, olfactory and respiratory microsomes from rat nasal mucosa preferentially activate NPIP over NPYR with P450 2A3 likely playing a key role. These results are consistent with local metabolic activation of nitrosamines as a contributing factor in their tissue-specific carcinogenicity.

Animals↗
PubMed1995

The role of the circle of Willis in carotid occlusion: assessment with phase contrast MR angiography and transcranial duplex.

PURPOSE: To study the collateral pathways recruited after occlusion of the internal carotid artery (ICA), and to evaluate its influence on the impairment of hemispheric blood flow supply and development of low flow infarcts. METHODS: 38 patients with ICA occlusion (18 asymptomatic; five transient ischaemic attacks; and 15 strokes) were included. Infarcts on cerebral MR scanning were categorised in order to differentiate patients with territorial infarcts or no lesion (group I; n = 22) from those with brain damage due to low flow (group II; n = 16). Patency and direction of flow in the communicating arteries were assessed by means of cine phase contrast MR angiography (PC-MRA). Flow velocity in the middle cerebral artery (MCA) was measured by means of transcranial Duplex (TCD). RESULTS: Cine PC-MRA revealed a reversed ophthalmic artery blood flow ipsilateral to the ICA occlusion in all except two patients in group I and one patient in group II (NS). Posterior to anterior flow in the ipsilateral posterior communicating artery (PCoA) was detected in 16 (73%) patients in group I and in 13 (81%) in group II (NS). In contrast, reversed blood flow in the ipsilateral A1 segment of the anterior cerebral artery, through a patent anterior communicating artery (ACoA), was identified in 19 (86%) patients of group I, vs. 7 (44%) of group II (p = 0.005). The relative risk of low-flow infarcts was significantly higher in those cases with non-functioning ACoA (odds ratio = 8.1; p < 0.05). TCD showed a lower peak systolic velocity (PSV) in the ipsilateral MCA than in the contralateral one (60 +/- 9 cm/s vs. 90 +/- 11 cm/s; p < 0.005). Those patients without crossed flow through the ACoA, showed an even lower PSV in the ipsilateral MCA (55 +/- 7 cm/s vs. 64 +/- 9 cm/s; p = 0.03). CONCLUSIONS: These data suggest that even though ICA occlusion may occur without cerebral damage, collateral blood supply is not enough to maintain normal hemispheric perfusion. The ACoA may be a key collateral pathway as a non-functioning ACoA is associated with an increased risk of developing low-flow infarcts.

Adolescent↗
PubMed2005

Analysis of flowering pathway integrators in Arabidopsis.

Flowering is regulated by an integrated network of several genetic pathways in Arabidopsis. The key genes integrating multiple flowering pathways are FT, SOC1 and LFY. To elucidate the interactions among these integrators, genetic analyses were performed. FT and SOC1 share the common upstream regulators CO, a key component in the long day pathway, and FLC, a flowering repressor integrating autonomous and vernalization pathways. However, the soc1 mutation further delayed the flowering time of long day pathway mutants including ft, demonstrating that SOC1 acts partially independently of FT. Although soc1 did not show an obvious defect in flower meristem determination on its own, it dramatically increased the number of coflorescences in a lfy mutant, which is indicative of a defect in floral initiation. Therefore, double mutant analysis shows that the three integrators have both overlapping and independent functions in the determination of flowering time and floral initiation. The expression analysis showed that FT regulates SOC1 expression, and SOC1 regulates LFY expression, but not vice versa, which is consistent with the fact that FT and LFY have the least overlapping functions among the three integrators. The triple mutation ft soc1 lfy did not block flowering completely under long days, indicating the presence of other integrators. Finally, vernalization accelerated flowering of flc ft soc1 and ft soc1 lfy triple mutants, which shows that the vernalization pathway also has targets other than FLC, FT, SOC1 and LFY. Our genetic analysis reveals the intricate nature of genetic networks for flowering.

Arabidopsis↗