ACTA PHYSIOLOGICA, Issue 3 2010
G. Flemström
Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source]

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis

CYTOSKELETON, Issue 6 2008
Amirali Sattarzadeh
Abstract Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens -based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

Molecular cloning and sequence analysis of an ascidian egg ,-N-acetylhexosaminidase with a potential role in fertilization

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2003
Ryo Koyanagi
,-N-Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm,egg coat binding. In ascidians and mammals, ,-hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of ,-hexosaminidase. In the present study, P. mammillata,-hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known ,-hexosaminidases; however, P. mammillata,-hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata,-hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of ,-hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg ,-hexosaminidase forms are specific for the oocyte, test cells and follicle cells. [source]

Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000
Kazuo Tomita
The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source]

Functional retinoid receptors in budding ascidians

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2000
Mika Kamimura
A homolog of retinoid X receptors (RXR), named PmRXR, was cloned from the budding ascidian, Polyandrocarpa misakiensis. Gel-shift assays revealed that PmRXR and a previously identified P. misakiensis retinoic acid receptor (PmRAR) formed a complex to bind vertebrate-type retinoic acid response element (RARE). Transfection assays were carried out using a reporter gene containing a RARE upstream of lacZ. Two chimeric effector genes were constructed by placing PmRXR and PmRAR cDNA fragments (containing the DNA-binding, ligand-binding and ligand-dependent transactivation domains) downstream of the human RXR, and RAR, cDNA (covering the N-terminal coding region), respectively. Each chimeric cDNA was ligated to a notochord-specific enhancer. In case the embryos were transfected with all three transgenes and treated with retinoic acid (RA), the reporter gene was activated in the notochord cells. The result suggests that the PmRXR/PmRAR complex functions as an RA-dependent transcriptional activator. The PmRXR mRNA was detected in a mesenchymal cell type, called glomerulocyte, in the developing Polyandrocarpa bud. As this cell type has been shown to express PmRAR mRNA, it seems possible that the PmRXR/PmRAR complex mediates RA signaling in this cell type to induce the expression of genes involved in the morphogenesis of the developing bud. [source]

Expression of a novel zebrafish zinc finger gene, gli2b, is affected in Hedgehog and Notch signaling related mutants during embryonic development

DEVELOPMENTAL DYNAMICS, Issue 2 2005
Zhiyuan Ke
Abstract Gli zinc-finger proteins are known as downstream mediators of the evolutionary conserved Hedgehog pathway. In zebrafish, gli2 functions differently from Gli2 in mammals. This difference could be due to the gli2 duplication in teleosts evolution and partial redundancy between two duplicated genes. Here, we report a novel zebrafish gli2 -like cDNA. Its structure, genetic location, and distinct expression pattern in the central nervous system suggested that this gene might represent a second gli2 of teleosts, and we named it gli2b. gli2b was expressed in the neural keel, excluding the forebrain,midbrain boundary, while gli2 expression complemented this pattern. After 24 hours postfertilization, several specific domains of gli2b expression were observed in the lateral and medial hindbrain and hypothalamus. In mutants affecting the Hedgehog and Notch signaling pathways, gli2b expression was either disrupted or extended in different regions. Developmental Dynamics 232:479,486, 2005. © 2005 Wiley-Liss, Inc. [source]

Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouse

DEVELOPMENTAL DYNAMICS, Issue 4 2003
Reginald Van Wayenbergh
Abstract Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity. Developmental Dynamics 228:716,725, 2003. © 2003 Wiley-Liss, Inc. [source]

Two Na,K-ATPase ,2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis

DEVELOPMENTAL DYNAMICS, Issue 2 2002
Johannes R. Rajarao
Abstract We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase ,2 subunit. The ,2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish ,2a subunit. By using a zebrafish meiotic mapping panel, we determined that the ,2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the ,2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. ,2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, ,2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the ,2a and ,2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian ,2 gene may be partitioned between the two zebrafish ,2 orthologs. © 2002 Wiley-Liss, Inc. [source]

Drosophila RSK negatively regulates bouton number at the neuromuscular junction

DEVELOPMENTAL NEUROBIOLOGY, Issue 4 2009
Matthias Fischer
Abstract Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, we investigated the effect of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk -cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source]

Expressed sequence tag analysis of the diapausing queen of the bumblebee Bombus ignitus

ENTOMOLOGICAL RESEARCH, Issue 4 2006
Yeon-Ju KIM
Abstract We constructed a full-length cDNA library from diapausing queens of the bumblebee Bombus ignitus. A total of 480 randomly selected clones was sequenced by single-run 5,-end sequencing. Of these, there were 437 high quality clones, 23 poor quality clones and 20 read-fail clones. Each high quality clone sequence was searched against a public protein database. The most frequently found matching genes were ribosomal proteins (12.5%), p10 (3.58%), cytochrome P450 monooxygenase (3.13%) and sensory appendage protein (2.9%). Sequence similarity analysis between bumblebees and other insect species showed that 72 out of 437 (16.5%) bumblebee expressed sequence tags (EST) matched sequences of Apis mellifera, with matches to Drosophila melanogaster (6.6%), Caenorhabditis briggsae (6.2%), Lysiphlebus testaceipes (4.8%), Periplaneta americana (3.7%) and Anopheles gambiae (3.4%) following, suggesting that sequence similarity of bumblebee EST is closest to that of A. mellifera. Functional classification of EST based on Gene Ontology showed that most genes found by sequencing are associated with physiological processes in the bumblebee. The results of sequencing and analysis of our 437 cDNA demonstrated that high-throughput EST sequencing and data analysis are powerful means for identifying novel genes and for expression profiling. Our bumblebee EST collection could be a useful platform for further studies of gene expression in diapausing bumblebees. [source]

Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2004
Quanxin Meng
Abstract A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C,C:G and A:T,T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5, partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats. Environ. Mol. Mutagen. 43:75,92, 2004. © 2004 Wiley-Liss, Inc. [source]

Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3,-azido-3,-deoxythymidine and 2,,3,-dideoxyinosine,

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2002
Quanxin Meng
Abstract Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3,-azido-2,,3,-dideoxythymidine (AZT) and 2,,3,-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667,12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 ,M or 300 ,M AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249,259; Meng Q et al. [2000b]: Toxicol Sci 54:322,329). Molecular analyses of HPRT mutant clones were performed by reverse transcription,mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 ,M AZT,ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency × percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 × 10,6 in HPRT and 6.0 × 10,6 in TK) at exposure levels of 100 ,M AZT,ddI (i.e., 1.94 × 10,6 in HPRT and 18.6 × 10,6 in TK) and 300 ,M AZT,ddI (i.e., 5.6 × 10,6 in HPRT and 34.6 × 10,6 in TK) (P < 0.05, Mann,Whitney U -statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 × 10,6) in cells exposed to 100 and 300 ,M AZT,ddI, respectively]. These results indicate that, at the same time that AZT,ddI potentiates therapeutic or prophylactic efficacy, the use of a second NRTI with AZT may confer a greater cancer risk, characterized by a spectrum of mutations that deviates from that produced solely by AZT. Environ. Mol. Mutagen. 39:282,295, 2002. Published 2002 Wiley-Liss, Inc. [source]

Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2007
Patrick Larkin
Abstract Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17,-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17,-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species. [source]

Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004
Jisung Ryu
Abstract The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by ,-naphthoflavone (,NF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription,polymerase chain reaction. The concentration,dependent induction of CYP1A mRNA in the liver was observed after exposure to ,NF at nominal concentrations of 20, 100, and 500 ,g/ L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 ,g/L of ,NF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to ,NF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the ,NF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals. [source]

Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2002
Gabriele E. Ackermann
Abstract This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtER, cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtER, cDNA expression vector, and clones responsive to 17,-estradiol (E2) were selected. The estrogenic activity of E2, 17,-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4- tert -octylphenol, bisphenol A, o,p,-DDT, p,p,-DDT, o,p,-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p,-DDE), p,p,-DDE, o,p,-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p,-DDD), p,p,-DDD, and p,p,-2,2-bis(chlorophenyl)acetic acid (p,p,-DDA) was assessed at increasing concentrations. All compounds except o,p,-DDT, p,p,-DDE, and p,p,-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p,-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed. [source]

Construction of a cDNA library of Bemisia tabaci for use in the ,yeast two-hybrid screen' method

EPPO BULLETIN, Issue 1 2002
S. Ohnesorge
The molecular mechanisms involved in the circulative, non-propagative transmission pathway of TYLCV through its vector the whitefly Bemisia tabaci have hardly been studied. Points requiring investigation include the specific adhesion of virus coat protein to insect structures, the proteins involved in membrane passage in the insect and the possibility of replication of the virus in the vector. To isolate the insect proteins which are involved in transmission by interaction with viral proteins, we propose to use the ,yeast two-hybrid screen' genetic method. For this method, it is indispensable to have a ,cDNA library' of the organism concerned, cloned in plasmids, and our first step has been to develop this. A new method was developed for isolating whitefly mRNA. From this mRNA, cDNA was synthesized, ligated in the plasmid pGADT7 (Clontech) and transformed in bacteria to amplify the plasmid DNA. The number of independent clones and average insert size of the plasmids were determined. [source]

Comparative genomics of the Mill family: a rapidly evolving MHC class,I gene family

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2004
Yutaka Watanabe
Abstract Mill (MHC class,I-like located near the leukocyte receptor complex) is a novel family of class,I genes identified in mice that is most closely related to the human MICA/B family. In the present study, we isolated Mill cDNA from rats and carried out a comparative genomic analysis. Rats have two Mill genes orthologous to mouse Mill1 and Mill2 near the leukocyte receptor complex, with expression patterns similar to those of their mouse counterparts. Interspecies sequence comparison indicates that Mill is one of the most rapidly evolving class,I gene families and that non-synonymous substitutions occur more frequently than synonymous substitutions in its ,,1 domain, implicating the involvement of Mill in immune defenses. Interestingly, the ,,2 domain of rat Mill2 contains a premature stop codon in many inbred strains, indicating that Mill2 is not essential for survival. A computer search of the database identified a horse Mill -like expressed sequence tag, indicating that Mill emerged before the radiation of mammals. Hence, the failure to find Mill in human indicates strongly that it was lost from the human lineage. Our present work provides convincing evidence that Mill is akin to the MICA/B family, yet constitutes a distinctgene family. [source]

Silencing dopamine D3 -receptors in the nucleus accumbens shell in vivo induces changes in cocaine-induced hyperlocomotion

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005
Amine Bahi
Abstract The dopamine D3 receptor (D3R) is an important pharmacotherapeutic target for its potential role in psychiatric disorders and drug dependence. To further explore its function in rats, a regulatable lentivirus, Lenti-D3, holding the rat D3R cDNA, has been constructed as well as three nonregulatable lentiviruses, Lenti-D3-siRNA1, Lenti-D3-siRNA2 and Lenti-D3-siRNA3, expressing small hairpin RNAs, aimed at silencing D3R expression and specifically targeted against different regions of the D3R mRNA. In vitro, Lenti-D3 expressed D3R and could efficiently be blocked with Lenti-D3-Sils. These viruses were stereotaxically injected into the shell part of the nucleus accumbens (NAcc) and effects of passive cocaine delivery on locomotor activity were assessed. Manipulations of D3R levels induced changes in the locomotor stimulant effects of cocaine as compared to control treatment. Suppression of dopamine (DA) D3R in the NAcc by means of local knockdown (with Lenti-D3-Sils) increased locomotor stimulant effects, whereas its overexpression with Lenti-D3 drastically reduced them. The latter effects could be reversed when animals were fed doxycycline, which prevented lentiviral-mediated DA D3R overexpression in the NAcc. Gene expression assessed by quantitative RT-PCR confirmed very efficient gene knockdown in vivo in animals treated with Lenti-D3-Sils (> 93% silencing of D3R gene). Thus D3R expression significantly contributes to behavioural changes associated with chronic cocaine delivery. [source]

Absence of phosphoglucose isomerase-1 in retinal photoreceptor, pigment epithelium and Muller cells

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004
Simon N. Archer
Abstract Macroarray analysis was used to compare equal amounts of cDNA from wild-type and rd/rd (retinal degeneration) mice, collected at P90 when photoreceptor degeneration is virtually complete. A stronger signal for the glycolytic enzyme phosphoglucose isomerase (Gpi1) was observed in the rd/rd sample. Extracellularly, Gpi1 may act as a cytokine, independently described as neuroleukin and autocrine motility factor. Retinal Gpi1 expression was investigated by Northern and Western blot analysis and immunohistochemistry. Double-labelling was performed with antibodies against Gpi1 and calbindin-D, glutamine synthetase, RPE65, calretinin and ultraviolet opsin in order to provide positive cell type identification. Northern and Western blots showed double expression levels per microgram of RNA and protein, respectively, in the rd/rd retina compared with wild-type. However, the total amount of Gpi1 protein per retina was indistinguishable. Gpi1 immunoreactivity was found in ganglion, amacrine, horizontal and bipolar cells, but not in rods, cones, pigment epithelium and Muller cells. This distribution explains why the absolute amounts of Gpi1 protein were not appreciably different between wild-type and the rd/rd phenotype, where rods and cones are absent, whilst the relative contribution of Gpi1 to the total protein and RNA pools differed. Some extracellular immunoreactivity was observed in the photoreceptor matrix around cones in freshly fixed tissue only, which could possibly reflect a role as a cytokine. We propose that glycolysis in Gpi1-negative cells proceeds entirely through the pentose phosphate pathway, creating NADPH at the cost of organic carbon. We hypothesize that the unique metabolic needs of photoreceptors justify this trade-off. [source]

Cloning and characterization of SDF-1,, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2000
Marc Gleichmann
Abstract The cytokines SDF-1, and -1, are two alternatively spliced variants of the CXC (,) chemokines that are highly conserved among species. SDF-1, was shown to function as a B-cell maturation factor, a ligand for the CXCR4 (LESTR/fusin) chemokine receptor, thereby inhibiting replication of T cell-tropic HIV-1 strains and inducing cell death in human neuronal cell lines. In this report the cloning of the rat SDF-1, cDNA and a new SDF-1 isoform, SDF-1,, are presented. Using Northern blot analysis, the expression pattern of both isoforms was studied in different tissues and it is shown that during postnatal development of the central and peripheral nervous system SDF-1,- and SDF-1,-mRNA expression is inversely regulated. Whilst SDF-1,-mRNA is the predominant isoform in embryonic and early postnatal nerve tissue, SDF-1,-mRNA is expressed at higher levels in adulthood. After peripheral nerve lesion a transient increase in SDF-1,-mRNA expression is observed. As revealed by in situ hybridization, neurons and Schwann cells are the main cellular sources of both SDF-1, and SDF-1, mRNAs in the nervous system. Computer-assisted analysis revealed that both transcripts encode secreted peptides with putative proteolytic cleavage sites which might generate novel neuropeptides. [source]

Genomic annotation and transcriptome analysis of the zebrafish (Danio rerio) hox complex with description of a novel member, hoxb13a

EVOLUTION AND DEVELOPMENT, Issue 5 2005
M. Corredor-Adámez
Summary The zebrafish (Danio rerio) is an important model in evolutionary developmental biology, and its study is being revolutionized by the zebrafish genome project. Sequencing is at an advanced stage, but annotation is largely the result of in silico analyses. We have performed genomic annotation, comparative genomics, and transcriptional analysis using microarrays of the hox homeobox-containing transcription factors. These genes have important roles in specifying the body plan. Candidate sequences were located in version Zv4 of the Ensembl genome database by TBLASTN searching with Danio and other vertebrate published Hox protein sequences. Homologies were confirmed by alignment with reference sequences, and by the relative position of genes along each cluster. RT-PCR using adult Tübingen cDNA was used to confirm annotations, to check the genomic sequence and to confirm expression in vivo. Our RT-PCR and microarray data show that all 49 hox genes are expressed in adult zebrafish. Significant expression for all known hox genes could be detected in our microarray analysis. We also find significant expression of hox8 paralogs and hoxb7a in the anti-sense direction. A novel gene, D. rerio hoxb13a, was identified, and a preliminary characterization by in situ hybridization showed expression at 24 hpf at the tip of the developing tail. We are currently characterizing this gene at the functional level. We argue that the oligo design for microarrays can be greatly enhanced by the availability of genomic sequences. [source]

Novel member of the mouse desmoglein gene family: Dsg1-,

EXPERIMENTAL DERMATOLOGY, Issue 1 2003
L. Pulkkinen
Abstract: Desmosomes are major intercellular adhesion junctions that provide stable cell,cell contacts and mechanical strength to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells. Desmogleins (Dsg) are transmembrane core components of the desmosomes, and belong to the cadherin supergene family of calcium-dependent adhesion molecules. Currently, there are three known isoforms of Dsgs (Dsg1, Dsg2, and Dsg3), encoded by distinct genes that are differentially expressed to determine their tissue specificity and differentiation state of epithelial cells. In this study, we cloned a novel mouse desmoglein gene sharing high homology to both mouse and human Dsg1. We propose to designate the previously published mouse Dsg1 gene as Dsg1- , and the new gene as Dsg1-,. Analysis of intron/exon organization of the Dsg1-, and Dsg1-, genes revealed significant conservation. The full-length mouse Dsg1-, cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids. Dsg1-, protein shares 94% and 76% identity with mouse Dsg1-, and human DSG1, respectively. RT-PCR using a multitissue cDNA panel demonstrated that while Dsg1-, mRNA was expressed in 15- to 17-day-old embryos and adult spleen and testis, Dsg1-, mRNA was detected in 17-day-old embryos only. To assess subcellular localization, a FLAG-tagged expression construct of Dsg1-, was transiently expressed in epithelial HaCaT cells. Dsg1-,-FLAG was found at the cell,cell border and was recognized by the anti-Dsg1/Dsg2 antibody DG3.10. In summary, we have cloned and characterized a novel member of the mouse desmoglein gene family, Dsg1-,. [source]

Characterization of Zebrafish Cx43.4 Connexin and its Channels

EXPERIMENTAL PHYSIOLOGY, Issue 6 2003
T. Desplantez
Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a Mr of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (Vj). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive Vj: Vj,0= -38.4/41.9 mV; gj,min= 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (,j,main= 33.2 ± 1.5 pS) and a residual conductance state (,j,residual= 11.9 ± 0.6 pS). [source]

MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene but post-translationally regulated by proteasome-dependent protein turnover

FEBS JOURNAL, Issue 20 2010
Jrhau Lung
The c-myc promoter-binding protein-1 (MBP-1) is a transcriptional suppressor of tumorigenesis and thought to be the product of alternative translation initiation of the ,-enolase (ENO1) transcript. In the present study, we cloned a 2552-bp novel cDNA with a putative coding sequence of MBP-1 and functionally examined its ability to encode the MBP-1 protein. Similarly to ENO1, the obtained MBP-1 was widely and differentially expressed in a variety of normal tissues and cancer cells. Experiments using MBP-1 promoter-driven luciferase reporter assays, biochemical cell fractionation followed by RT-PCR detection of the cytoplasmic mRNA, and transcription/translation-coupled reactions, consistently demonstrated that this novel transcript was alternatively transcribed from intron III of the ENO1 gene and was feasible for MBP-1 production. Hypoxia treatments significantly increased the transcriptional activation of the MBP-1 gene. Blocking the proteasomal degradation by MG132 stabilized the MBP-1 protein in cells. Compared with the translation efficiency for production of the MBP-1 protein, the MBP-1 transcript was 17.8 times more efficient than the ENO1 transcript. Thus, we suggest that this newly discovered transcript is a genuine template for the protein synthesis of MBP-1 in cells, and optimal expression of this gene in tumors may lead to effective clinical therapies for cancers. [source]

Catalytic digestion of human tumor necrosis factor-, by antibody heavy chain

FEBS JOURNAL, Issue 18 2010
Emi Hifumi
It has long been an important task to prepare a catalytic antibody capable of digesting a targeting crucial protein that controls specific life functions. Tumor necrosis factor-, (TNF-,) is a cytokine and an important molecule concerned with autoimmune diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn's disease. A mAb (ETNF-6 mAb) raised against human TNF-, was prepared, and the steric conformation was created by using molecular modeling after the cDNA was sequenced. The heavy chain (ETNF-6-H) of the mAb was considered to possess a catalytic triad-like structure in the complementarity determining regions (CDRs). As a result, ETNF-6-H exhibited a peptidase and a protease activity. In fact, ETNF-6-H predominantly cleaved the Ser5-Arg6 bond of TNF-, at the first step, resulting in the generation of a fragment of , 17 kDa. This fragment was digested to a smaller molecule of 15 kDa by scission of the Gln21-Ala22 bond. The intermediate product was further converted into a fragment of 13.3 kDa by successive cleavage of the Leu36-Leu37 and Asn39-Gly40 bonds. The heavy chain possessed a protease activity against TNF-, with a multicleavage site. [source]

Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activity

FEBS JOURNAL, Issue 6 2008
Ursula Theissen
The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mm. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (Km = 6.4 ,m) after the addition of sulfide (Km = 23 ,m) only in the presence of cyanide (Km = 2.6 mm). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme. [source]

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

FEBS JOURNAL, Issue 6 2008
Adam Frederick
To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChET subunit, which should be able to produce all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G1a) and non-amphiphilic tetramers (G4na) are found. Amphiphilic dimers (G2a) and non-amphiphilic tetramers (G4na) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A12 form of the enzyme. Collagenase digestion of the A12 AChE produces a lytic G4 form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates. [source]

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

FEBS JOURNAL, Issue 24 2007
Andre Müller
cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5,- and 3,-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH,5His was purified to homogeneity using metal,chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in Km and decreases in kcat values for pyruvate and l -arginine, but had little effect on the Km and kcat values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid,base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329. [source]

Identification of an osteopontin-like protein in fish associated with mineral formation

FEBS JOURNAL, Issue 17 2007
Vera G. Fonseca
Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates. [source]

Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus

FEBS JOURNAL, Issue 5 2007
Santosh Kumar
Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a ,three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a ,-glucuronidase,green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed. [source]