Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

The mitochondrial ornithine transporter. Bacterial expression,reconstitution, functional characterization,and tissue distribution of two human isoforms

Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1.     

Molecular interaction and enzymatic activity of macrophage migration inhibitory factor with immunorelevant peptides

Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP beta1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed.     

BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.     

Fixed precision sequential sampling plans for the greenbug and bird cherry-oat aphid (Homoptera: Aphididae) in winter wheat

The numbers of greenbugs, Schizaphis graminum (Rondani), and bird cherry-oat aphids, Rhopalosiphum padi L., per wheat tiller (stem) were estimated in 189 production winter wheat (Triticum aestivum L.) fields located throughout Oklahoma. Taylor's power law regressions were calculated from these data and used to construct fixed precision sequential sampling schemes for each species. An evaluation data set was constructed from 240 samples taken during three growing seasons from winter wheat fields at four locations in Oklahoma. Wheat cultivar and growth stage were recorded for each field on the day of sampling. Taylor's power law parameters for evaluation fields differed significantly for both species among growing seasons, locations, and plant growth stages. Median precision achieved using the fixed precision sequential sampling schemes for each species departed <20% from expected precision over the range population intensity in the evaluation data. For the 10% of samples with greatest deviation between observed and expected precision, observed precision was 13.8-81.8% greater than that expected precision depending on aphid species and population intensity. For the greenbug, the distribution of the percentage deviation between observed and expected precision was positively skewed, so that the sampling scheme tended to over-predict precision. For the bird cherry-oat aphid, the distribution was more symmetric. Even though precision observed using the sampling schemes frequently varied from expected precision, because of the inevitable consequence of sampling error and environmental variation, the sampling schemes yielded median observed precision levels close to expected precision levels over a broad range of population intensity.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

Bacterial host strains that support replication of somatic coliphages

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log₁₀ fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. This revised version was published online in June 2006 with corrections to the Cover Date.