Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for themore » first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.« less

Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular carcinomas.

PubMed

Fairchild, C R; Ivy, S P; Rushmore, T; Lee, G; Koo, P; Goldsmith, M E; Myers, C E; Farber, E; Cowan, K H

1987-11-01

We have previously reported the isolation of a human breast cancer cell line resistant to doxorubicin (adriamycin; AdrR MCF-7 cells) that has also developed the phenotype of multidrug resistance (MDR). MDR in this cell line is associated with increased expression of mdr (P glycoprotein) gene sequences. The development of MDR in AdrR MCF-7 cells is also associated with changes in the expression of several phase I and phase II drug-detoxifying enzymes. These changes are remarkably similar to those associated with development of xenobiotic resistance in rat hyperplastic liver nodules, a well-studied model system of chemical carcinogenesis. Using an mdr-encoded cDNA sequence isolated from AdrR MCF-7 cells, we have examined the expression of mdr sequences in rat livers under a variety of experimental conditions. The expression of mdr increased 3-fold in regenerating liver. It was also elevated (3- to 12-fold) in several different samples of rat hyperplastic nodules and in four of five hepatomas that developed in this system. This suggests that overexpression of mdr, a gene previously associated with resistance to antineoplastic agents, may also be involved in the development of resistance to xenobiotics in rat hyperplastic nodules. In addition, although the acute administration of 2-acetylaminofluorene induced an 8-fold increase in hepatic mdr-encoded RNA, performance of a partial hepatectomy either before or after administration of 2-acetylaminofluorene resulted in a greater than 80-fold increase in mdr gene expression over that in normal untreated livers. This represents an important in vivo model system in which to study the acute regulation of this drug resistance gene.

Effect of resveratrol analogue, DMU-212, on antioxidant status and apoptosis-related genes in rat model of hepatocarcinogenesis.

PubMed

Piotrowska, H; Kujawska, M; Nowicki, M; Petzke, E; Ignatowicz, E; Krajka-Kuźniak, V; Zawierucha, P; Wierzchowski, M; Murias, M; Jodynis-Liebert, J

2017-02-01

The aim of the study was to examine whether antioxidant properties of 3,4,4',5-tetramethoxystilbene (DMU-212) contribute to its anticarcinogenic activity and whether DMU-212 affects the expression of apoptosis-related genes. Two-stage model of hepatocarcinogenesis was used; male Wistar rats were challenged with N-nitrosodiethylamine (NDEA), 200 mg/kg body weight (b.w.), intraperitoneal, then phenobarbital (PB) in drinking water (0.05%) was administered. Simultaneously, DMU-212 was given per os at a dose 20 or 50 mg/kg b.w. two times a week for 16 weeks. DMU-212 caused a moderate decrease in hepatic thiobarbituric acid reactive substances and protein carbonyls concentration elevated in rats treated with NDEA/PB. The activity of antioxidant enzymes examined reduced by NDEA/PB treatment was not restored in rats coadministered with DMU-212. Effects of DMU-212 on messenger RNA (mRNA) expression of antioxidant enzymes in rats challenged with NDEA/PB were diversified; no changes in their protein expression were noted in any of the groups. The expression of 17,000 genes was analyzed by Affymetrix® Rat Gene 1.1 ST Array; 15 apoptosis-related genes were selected and validated by RT-q PCR. The combined treatment with NDEA/PB and DMU-212 increased the mRNA level of some genes driving mitochondria-mediated apoptosis, whereas the mRNA expression of some anti-apoptotic genes triggering receptor-mediated apoptosis was reduced. The expression of genes encoding caspases-4, -8, -9, and -12 was also increased in rats treated with DMU-212. Although antioxidant effect of DMU-212 in rats challenged with NDEA/PB was moderate, its potential anticarcinogenic properties were demonstrated as evidenced by modulation of apoptosis-related genes.

Transcriptional analysis of the bglP gene from Streptococcus mutans.

PubMed

Cote, Christopher K; Honeyman, Allen L

2006-04-21

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.

Transcriptional analysis of the bglP gene from Streptococcus mutans

PubMed Central

Cote, Christopher K; Honeyman, Allen L

2006-01-01

Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript. PMID:16630357

Screening a yeast promoter library leads to the isolation of the RP29/L32 and SNR17B/RPL37A divergent promoters and the discovery of a gene encoding ribosomal protein L37.

PubMed

Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

1991-08-30

Two promoters (A7 and A23), isolated at random from the Saccharomyces cerevisiae genome by virtue of their capacity to activate transcription, are identical to known intergenic bidirectional promoters. Sequence analysis of the genomic DNA adjacent to the A7 promoter identified a split gene encoding ribosomal (r) protein L37, which is homologous to the tRNA-binding r-proteins, L35a (from human and rat) and L32 (from frogs).

A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock.

PubMed

Okuyama, Tetsuya; Nakatake, Richi; Kaibori, Masaki; Okumura, Tadayoshi; Kon, Masanori; Nishizawa, Mikio

2018-01-30

Natural antisense transcripts (asRNAs) that do not encode proteins are transcribed from rat, mouse, and human genes, encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide (NO). In septic shock, NO is excessively produced in hepatocytes and macrophages. The iNOS asRNA interacts with and stabilizes iNOS mRNA. We found that single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduced iNOS mRNA levels by interfering with the mRNA-asRNA interactions in rat hepatocytes. The iNOS sense oligonucleotides that were substituted with phosphorothioate bonds and locked nucleic acids efficiently decreased the levels of iNOS mRNA and iNOS protein. In this study, the gene expression patterns in the livers of two endotoxemia model rats with acute liver failure were compared. Next, we optimized the sequence and modification of the iNOS sense oligonucleotides in interleukin 1β-treated rat hepatocytes. When a sense oligonucleotide was simultaneously administered with d-galactosamine and bacterial lipopolysaccharide (LPS) to rats, their survival rate significantly increased compared to the rats administered d-galactosamine and LPS alone. In the livers of the sense oligonucleotide-administered rats, apoptosis in the hepatocytes markedly decreased. These results suggest that natural antisense transcript-targeted regulation technology using iNOS sense oligonucleotides may be used to treat human inflammatory diseases, such as sepsis and septic shock. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of muscular dystrophy model rats with a CRISPR/Cas system.

PubMed

Nakamura, Katsuyuki; Fujii, Wataru; Tsuboi, Masaya; Tanihata, Jun; Teramoto, Naomi; Takeuchi, Shiho; Naito, Kunihiko; Yamanouchi, Keitaro; Nishihara, Masugi

2014-07-09

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

Engineering brown fat into skeletal muscle using ultrasound-targeted microbubble destruction gene delivery in obese Zucker rats: Proof of concept design.

PubMed

Bastarrachea, Raul A; Chen, Jiaxi; Kent, Jack W; Nava-Gonzalez, Edna J; Rodriguez-Ayala, Ernesto; Daadi, Marcel M; Jorge, Barbara; Laviada-Molina, Hugo; Comuzzie, Anthony G; Chen, Shuyuan; Grayburn, Paul A

2017-09-01

Ultrasound-targeted microbubble destruction (UTMD) is a novel means of tissue-specific gene delivery. This approach systemically infuses transgenes precoupled to gas-filled lipid microbubbles that are burst within the microvasculature of target tissues via an ultrasound signal resulting in release of DNA and transfection of neighboring cells within the tissue. Previous work has shown that adenovirus containing cDNA of UCP-1, injected into the epididymal fat pads in mice, induced localized fat depletion, improving glucose tolerance, and decreasing food intake in obese diabetic mice. Our group recently demonstrated that gene therapy by UTMD achieved beta cell regeneration in streptozotocin (STZ)-treated mice and baboons. We hypothesized that gene therapy with BMP7/PRDM16/PPARGC1A in skeletal muscle (SKM) of obese Zucker diabetic fatty (fa/fa) rats using UTMD technology would produce a brown adipose tissue (BAT) phenotype with UCP-1 overexpression. This study was designed as a proof of concept (POC) project. Obese Zucker rats were administered plasmid cDNA contructs encoding a gene cocktail with BMP7/PRDM16/PPARGC1A incorporated within microbubbles and intravenously delivered into their left thigh. Controls received UTMD with plasmids driving a DsRed reporter gene. An ultrasound transducer was directed to the thigh to disrupt the microbubbles within the microcirculation. Blood samples were drawn at baseline, and after treatment to measure glucose, insulin, and free fatty acids levels. SKM was harvested for immunohistochemistry (IHC). Our IHC results showed a reliable pattern of effective UTMD-based gene delivery in enhancing SKM overexpression of the UCP-1 gene. This clearly indicates that our plasmid DNA construct encoding the gene combination of PRDM16, PPARGC1A, and BMP7 reprogrammed adult SKM tissue into brown adipose cells in vivo. Our pilot established POC showing that the administration of the gene cocktail to SKM in this rat model of genetic obesity using UTMD gene therapy, engineered a BAT phenotype with UCP-1 over-expression. © 2017 IUBMB Life, 69(9):745-755, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

A novel gene, RSD-3/HSD-3.1, encodes a meiotic-related protein expressed in rat and human testis.

PubMed

Zhang, Xiaodong; Liu, Huixian; Zhang, Yan; Qiao, Yuan; Miao, Shiying; Wang, Linfang; Zhang, Jianchao; Zong, Shudong; Koide, S S

2003-06-01

The expression of stage-specific genes during spermatogenesis was determined by isolating two segments of rat seminiferous tubule at different stages of the germinal epithelium cycle delineated by transillumination-delineated microdissection, combined with differential display polymerase chain reaction to identify the differential transcripts formed. A total of 22 cDNAs were identified and accepted by GenBank as new expressed sequence tags. One of the expressed sequence tags was radiolabeled and used as a probe to screen a rat testis cDNA library. A novel full-length cDNA composed of 2228 bp, designated as RSD-3 (rat sperm DNA no.3, GenBank accession no. AF094609) was isolated and characterized. The reading frame encodes a polypeptide consisting of 526 amino acid residues, containing a number of DNA binding motifs and phosphorylation sites for PKC, CK-II, and p34cdc2. Northern blot of mRNA prepared from various tissues of adult rats showed that RSD-3 is expressed only in the testis. The initial expression of the RSD-3 gene was detected in the testis on the 30th postnatal day and attained adult level on the 60th postnatal day. Immunolocalization of RSD-3 in germ cells of rat testis showed that its expression is restricted to primary spermatocytes, undergoing meiosis division I. A human testis homologue of RSD-3 cDNA, designated as HSD-3.1 (GenBank accession no. AF144487) was isolated by screening the Human Testis Rapid-Screen arrayed cDNA library panels by RT-PCR. The exon-intron boundaries of HSD-3.1 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The cDNA consisted of 12 exons that span approximately 52.8 kb of the genome sequence and was mapped to chromosome 14q31.3.

Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells.

PubMed

Bajenova, O V; Zimmer, R; Stolper, E; Salisbury-Rowswell, J; Nanji, A; Thomas, P

2001-08-17

Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein (hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds (125)I-labeled CEA in a Ca(2+)-dependent fashion. Transfection of rat hnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells.

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Hepatic expression of spermatogenic genes and their transiently remarkable downregulations in Wistar-Kyoto rats in response to lead-nitrate administration: strain-difference in the gene expression patterns.

PubMed

Nemoto, Kiyomitsu; Ito, Sei; Yoshida, Chiaki; Miyata, Misaki; Kojima, Misaki; Degawa, Masakuni

2011-06-01

Administration of lead ion (Pb) to rats and mice affects hepatic functions such as the induction of hepatic cell proliferation and upregulation of cholesterol biosynthesis. To identify the genes for which expression changes in response to Pb-administration, we analyzed hepatic gene expression patterns in stroke-prone spontaneously hypertensive rat (SHRSP), its normotensive control, Wistar-Kyoto rat (WKY), and Spraque-Dawley (SD) rat strains, 3, 6, and 12 hr later after single i.v. injection of lead nitrate (LN) at a dose of 100 µmol using a DNA microarray technique. The data analysis demonstrated that the expression of a great number of genes was transiently and remarkably downregulated 3 hr after LN-injection, and then recovered to control levels only in LN-injected WKY. These normal hepatic expression levels in WKY and SHRSP were much higher than those in SD rats. Furthermore, most of these genes were ones thought to be expressed specifically in the spermatids and/or testes; i.e. genes encoding protamin 1, transition protein 1, and transition protein 2. These findings suggest that the regulation system common to expression of all of these genes could be a target site of Pb-toxic action, at least, in the liver of WKY, and that this system might be similar to the system essential for spermatogenesis, especially spermiogenesis, in the testis. In addition, it appears that clarifying the cause of the difference between the systems of WKY and SHRSP might aid in identifying the pathologic genes in SHRSP. Finally, it will be an important to clarify how the products of the genes related to spermatogenesis, including spermiogenesis, are functional in the livers of WKY and SHRSP.

AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

PubMed

Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

2014-10-30

For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

A Brain Membrane Protein Similar to the Rat src Gene Product

NASA Astrophysics Data System (ADS)

Scheinberg, David A.; Strand, Mette

1981-01-01

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 μ M). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

PubMed

Vollrath, D; Feng, W; Duncan, J L; Yasumura, D; D'Cruz, P M; Chappelow, A; Matthes, M T; Kay, M A; LaVail, M M

2001-10-23

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Regulation of gene expression in the mammalian eye and its relevance to eye disease.

PubMed

Scheetz, Todd E; Kim, Kwang-Youn A; Swiderski, Ruth E; Philp, Alisdair R; Braun, Terry A; Knudtson, Kevin L; Dorrance, Anne M; DiBona, Gerald F; Huang, Jian; Casavant, Thomas L; Sheffield, Val C; Stone, Edwin M

2006-09-26

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

The Guinea Pig as a Model for Sporadic Alzheimer’s Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

PubMed Central

Ong, Daniel; Wijaya, Linda; Laws, Simon M.; Taddei, Kevin; Newman, Morgan; Lardelli, Michael; Martins, Ralph N.; Verdile, Giuseppe

2013-01-01

We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes. PMID:23805206

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus.

PubMed Central

Raibaud, A; Zalacain, M; Holt, T G; Tizard, R; Thompson, C J

1991-01-01

Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411. Images PMID:2066341

Mutation in the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct5) gene causes autosomal recessive mutilating sensory neuropathy with spastic paraplegia.

PubMed

Bouhouche, A; Benomar, A; Bouslam, N; Chkili, T; Yahyaoui, M

2006-05-01

Mutilating sensory neuropathy with spastic paraplegia is a very rare disease with both autosomal dominant and recessive modes of inheritance. We previously mapped the locus of the autosomal recessive form to a 25 cM interval between markers D5S2048 and D5S648 on chromosome 5p. In this candidate interval, the Cct5 gene encoding the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (CCT) was the most obvious candidate gene since mutation in the Cct4 gene encoding the CCT delta subunit has been reported to be associated with autosomal recessive mutilating sensory neuropathy in mutilated foot (mf) rat mutant. A consanguineous Moroccan family with four patients displaying mutilating sensory neuropathy associated with spastic paraplegia was investigated. To identify the disease causing gene, the 11 coding exons of the Cct5 gene were screened for mutations by direct sequencing in all family members including the four patients, parents, and six at risk relatives. Sequence analysis of the Cct5 gene revealed a missense A492G mutation in exon 4 that results in the substitution of a highly conserved histidine for arginine amino acid 147. Interestingly, R147 was absent in 384 control matched chromosomes tested. This is the first disease causing mutation that has been identified in the human CCT subunit genes; the mf rat mutant could serve as an animal model for studying these chaperonopathies.

Zygote arrest 1 (Zar1) is an evolutionarily conserved gene expressed in vertebrate ovaries.

PubMed

Wu, Xuemei; Wang, Pei; Brown, Christopher A; Zilinski, Carolyn A; Matzuk, Martin M

2003-09-01

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. In mice, the Zar1 mRNA is detected as a 1.4-kilobase (kb) transcript that is synthesized exclusively in growing oocytes. To further understand the functions of ZAR1, we have cloned the orthologous Zar1 cDNA and/or genes for mouse, rat, human, frog, zebrafish, and pufferfish. The entire mouse Zar1 gene and a related pseudogene span approximately 4.0 kb, contain four exons, and map to adjacent loci on mouse chromosome 5. The human ZAR1 orthologous gene similarly consists of four exons and resides on human chromosome 4p12, which is syntenic with the mouse Zar1 chromosomal locus. Rat (Rattus norvegicus) and pufferfish (Fugu rubripes) Zar1 genes were recognized by database mining and deduced protein alignment analysis. The rat Zar1 gene also maps to a region that is syntenic with the mouse Zar1 gene locus on rat chromosome 14. Frog (Xenopus laevis) and zebrafish (Danio rerio) Zar1 orthologs were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends analysis of ovarian mRNA. Unlike mouse and human, the frog Zar1 is detected in multiple tissues, including lung, muscle, and ovary. The Zar1 mRNA appears in the cytoplasm of oocytes and persists until the tailbud stage during frog embryogenesis. Mouse, rat, human, frog, zebrafish, and pufferfish Zar1 genes encode proteins of 361, 361, 424, 295, 329, and 320 amino acids, respectively, and share 50.8%-88.1% amino acid identity. Regions of the N-termini of these ZAR1 orthologs show high sequence identity among these various proteins. However, the C-terminal 103 amino acids of these proteins, encoded by exons 2-4, contain an atypical eight-cysteine Plant Homeo Domain motif and are highly conserved, sharing 80.6%-98.1% identity among these species. These findings suggest that the carboxyl-termini of these ZAR1 proteins contain an important functional domain that is conserved through vertebrate evolution and that may be necessary for normal female reproduction in the transition from oocyte to embryonic life.

Fmr1 and Nlgn3 knockout rats: novel tools for investigating autism spectrum disorders.

PubMed

Hamilton, Shannon M; Green, Jennie R; Veeraragavan, Surabi; Yuva, Lisa; McCoy, Aaron; Wu, Yumei; Warren, Joe; Little, Lara; Ji, Diana; Cui, Xiaoxia; Weinstein, Edward; Paylor, Richard

2014-04-01

Animal models are critical for gaining insights into autism spectrum disorder (ASD). Despite their apparent advantages to mice for neural studies, rats have not been widely used for disorders of the human CNS, such as ASD, for the lack of convenient genome manipulation tools. Here we describe two of the first transgenic rat models for ASD, developed using zinc-finger nuclease (ZFN) methodologies, and their initial behavioral assessment using a rapid juvenile test battery. A syndromic and nonsyndromic rat model for ASD were created as two separate knockout rat lines with heritable disruptions in the genes encoding Fragile X mental retardation protein (FMRP) and Neuroligin3 (NLGN3). FMRP, a protein with numerous proposed functions including regulation of mRNA and synaptic protein synthesis, and NLGN3, a member of the neuroligin synaptic cell-adhesion protein family, have been implicated in human ASD. Juvenile subjects from both knockout rat lines exhibited abnormalities in ASD-relevant phenotypes including juvenile play, perseverative behaviors, and sensorimotor gating. These data provide important first evidence regarding the utility of rats as genetic models for investigating ASD-relevant genes.

Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

PubMed Central

Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

2017-01-01

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

Social and novel contexts modify hippocampal CA2 representations of space

PubMed Central

Alexander, Georgia M.; Farris, Shannon; Pirone, Jason R.; Zheng, Chenguang; Colgin, Laura L.; Dudek, Serena M.

2016-01-01

The hippocampus supports a cognitive map of space and is critical for encoding declarative memory (who, what, when and where). Recent studies have implicated hippocampal subfield CA2 in social and contextual memory but how it does so remains unknown. Here we find that in adult male rats, presentation of a social stimulus (novel or familiar rat) or a novel object induces global remapping of place fields in CA2 with no effect on neuronal firing rate or immediate early gene expression. This remapping did not occur in CA1, suggesting this effect is specific for CA2. Thus, modification of existing spatial representations might be a potential mechanism by which CA2 encodes social and novel contextual information. PMID:26806606

Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

PubMed

Nagata, K; Ogino, M; Shimada, M; Miyata, M; Gonzalez, F J; Yamazoe, Y

1999-02-15

A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats. Copyright 1999 Academic Press.

Central leptin regulates heart lipid content by selectively increasing PPAR β/δ expression.

PubMed

Mora, Cristina; Pintado, Cristina; Rubio, Blanca; Mazuecos, Lorena; López, Virginia; Fernández, Alejandro; Salamanca, Aurora; Bárcena, Brenda; Fernández-Agulló, Teresa; Arribas, Carmen; Gallardo, Nilda; Andrés, Antonio

2018-01-01

The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d ) and their target genes, adipose triglyceride lipase (encoded by Pnpla2 , herefater referred to as Atgl ), hormone sensitive lipase (encoded by Lipe , herefater referred to as Hsl ), pyruvate dehydrogenase kinase 4 ( Pdk4 ) and acyl CoA oxidase 1 ( Acox1 ), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 ( Scd1 ) and diacylglycerol acyltransferase 1 ( Dgat1 ) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight. © 2018 Society for Endocrinology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pestov, Nikolay B., E-mail: korn@mail.ibch.ru; Dmitriev, Ruslan I.; Kostina, Maria B.

Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2),more » the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.« less

De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia

PubMed Central

Gauthier, Julie; Champagne, Nathalie; Lafrenière, Ronald G.; Xiong, Lan; Spiegelman, Dan; Brustein, Edna; Lapointe, Mathieu; Peng, Huashan; Côté, Mélanie; Noreau, Anne; Hamdan, Fadi F.; Addington, Anjené M.; Rapoport, Judith L.; DeLisi, Lynn E.; Krebs, Marie-Odile; Joober, Ridha; Fathalli, Ferid; Mouaffak, Fayçal; Haghighi, Ali P.; Néri, Christian; Dubé, Marie-Pierre; Samuels, Mark E.; Marineau, Claude; Stone, Eric A.; Awadalla, Philip; Barker, Philip A.; Carbonetto, Salvatore; Drapeau, Pierre; Rouleau, Guy A.

2010-01-01

Schizophrenia likely results from poorly understood genetic and environmental factors. We studied the gene encoding the synaptic protein SHANK3 in 285 controls and 185 schizophrenia patients with unaffected parents. Two de novo mutations (R1117X and R536W) were identified in two families, one being found in three affected brothers, suggesting germline mosaicism. Zebrafish and rat hippocampal neuron assays revealed behavior and differentiation defects resulting from the R1117X mutant. As mutations in SHANK3 were previously reported in autism, the occurrence of SHANK3 mutations in subjects with a schizophrenia phenotype suggests a molecular genetic link between these two neurodevelopmental disorders. PMID:20385823

Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

PubMed

Pietrowski, D; Durante, M J; Liebstein, A; Schmitt-John, T; Werner, T; Graw, J

1994-07-08

The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

PubMed Central

Miyazato, Minoru; Sugaya, Kimio; Goins, William F.; Goss, James R.; Chancellor, Michael B.; de Groat, William C.; Glorioso, Joseph C.; Yoshimura, Naoki

2010-01-01

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 Kd form of the glutamic acid decarboxylase (GAD67) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in spinal cord injury (SCI) rats. One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. SCI rats without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40–45% and 38–40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD67 mRNA and protein levels were significantly increased in L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO following SCI predominantly via activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for the treatment of neurogenic DO. PMID:19225548

Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas.

PubMed

D'Amico, Agata Grazia; Maugeri, Grazia; Reitano, Rita; Cavallaro, Sebastiano; D'Agata, Velia

2016-10-01

PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

POM-ZP3, a bipartite transcript derived from human ZP3 and a POM121 homologue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kipersztok, S.; Osawa, G.A.; Liang, L.F.

1995-01-20

Human POM-ZP3 is a novel bipartite RNA transcript that is derived from a gene homologous to rat POM121 (a nuclear pore membrane protein) and ZP3 (a sperm receptor ligand in the zona pellucida). The 5{prime} region is 77% identical to the 5{prime} end of the coding region of rat POM121 and appears to represent a partial duplication of a gene encoding a human homologue of this rodent gene. The 3{prime} end of the POM-ZP3 transcript is 99% identical to ZP3 and appears to have arisen from a duplication of the last four exons (exons 5-8) of ZP3. Using Northern blotsmore » and RT-PCR, POM-ZP3 transcripts were detected in human ovaries, testes, spleen, thymus, lymphocytes, prostate, and intestines. The longest open reading frame encodes a conceptual protein of 210 amino acids, the first 76 of which are 83% identical to residues 241-315 of rat POM121. The next 125 amino acids are 98% identical to residues 239-363 of the 424-amino-acid human ZP3 protein. By fluorescence in situ hybridization, genomic fragments of ZP3 and a human homologue of POM121 were localized to chromosome 7q11.23. Taken together, these data suggest that partial duplications of human ZP3 and a POM121-like gene have resulted in a fusion transcript, POM-ZP3, that is expressed in multiple human tissues. 24 refs., 5 figs.« less

Regulation of gene expression in the mammalian eye and its relevance to eye disease

PubMed Central

Scheetz, Todd E.; Kim, Kwang-Youn A.; Swiderski, Ruth E.; Philp, Alisdair R.; Braun, Terry A.; Knudtson, Kevin L.; Dorrance, Anne M.; DiBona, Gerald F.; Huang, Jian; Casavant, Thomas L.; Sheffield, Val C.; Stone, Edwin M.

2006-01-01

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease. PMID:16983098

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Glutathione Modulates Recominant Rat Arsenic (+3 Oxidation State) Methyltransferase-Catalyzed Formation of Trimethylarsine Oxide and Trimethylarsine

EPA Science Inventory

Humans and other species enzymatically convert inorganic arsenic (iAs) into methylated metabolites. Although the major metabolites are mono- and dimethylated arsenicals, trimethylated arsenicals have been detected in urine following exposure to iAs. The AS3MT gene encodes an ars...

Disruption of the zinc metabolism in rat fœtal brain after prenatal exposure to cadmium.

PubMed

Ben Mimouna, Safa; Boughammoura, Sana; Chemek, Marouane; Haouas, Zohra; Banni, Mohamed; Messaoudi, Imed

2018-04-25

This study was carried out to investigate the effects of maternal Cd and/or Zn exposure on some parameters of Zn metabolism in fetal brain of Wistar rats. Thus, female controls and other exposed by the oral route during the gestation period to Cd (50 mg CdCl 2 /L) and/or Zn (ZnCl 2 60 mg/L) were used. The male fetuses at age 20 days of gestation (GD20) were sacrificed and their brains were taken for histological, chemical and molecular analysis. Zn depletion was observed in the brains of fetuses issued from mothers exposed to Cd. Histological analysis showed that Cd exposure induces pyknosis in cortical region and CA1 region of the hippocampus compared to controls. Under Cd exposure, we noted an overexpression of the genes coding for membrane transporter involved in the intracellular incorporation of Zn (ZIP6) associated with inhibition of that encoding the transporters involved in the output of the Zn into the extracellular medium (ZnT1 and ZnT3). A decrease in the expression of the gene encoding the neuro-trophic factor (BDNF) associated with overexpression of the encoding the metal regulatory transcription factor 1 (MTF1), factor involved in the homeostasis of Zn, was also noted in Cd group. Interestingly, Zn supply provided a total or partial restauration of the changes induced by the Cd exposure. The depletion of brain Zn contents as well as the modification of the profile of expression of genes encoding membrane Zn transporters, suggest that the toxicity of Cd observed in fetal brain level are mediated, in part, by impairment of Zn metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

PubMed Central

2010-01-01

Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus. PMID:20105316

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

A rat model of hypohidrotic ectodermal dysplasia carries a missense mutation in the Edaradd gene

PubMed Central

2011-01-01

Background Hypohidrotic ectodermal dysplasia (HED) is a congenital disorder characterized by sparse hair, oligodontia, and inability to sweat. It is caused by mutations in any of three Eda pathway genes: ectodysplasin (Eda), Eda receptor (Edar), and Edar-associated death domain (Edaradd), which encode ligand, receptor, and intracellular adaptor molecule, respectively. The Eda signaling pathway activates NF-κB, which is central to ectodermal differentiation. Although the causative genes and the molecular pathway affecting HED have been identified, no curative treatment for HED has been established. Previously, we found a rat spontaneous mutation that caused defects in hair follicles and named it sparse-and-wavy (swh). Here, we have established the swh rat as the first rat model of HED and successfully identified the swh mutation. Results The swh/swh rat showed sparse hair, abnormal morphology of teeth, and absence of sweat glands. The ectoderm-derived glands, meibomian, preputial, and tongue glands, were absent. We mapped the swh mutation to the most telomeric part of rat Chr 7 and found a Pro153Ser missense mutation in the Edaradd gene. This mutation was located in the death domain of EDARADD, which is crucial for signal transduction and resulted in failure to activate NF-κB. Conclusions These findings suggest that swh is a loss-of-function mutation in the rat Edaradd and indicate that the swh/swh rat would be an excellent animal model of HED that could be used to investigate the pathological basis of the disease and the development of new therapies. PMID:22013926

Identification and characterisation of the angiotensin converting enzyme-3 (ACE3) gene: a novel mammalian homologue of ACE

PubMed Central

Rella, Monika; Elliot, Joann L; Revett, Timothy J; Lanfear, Jerry; Phelan, Anne; Jackson, Richard M; Turner, Anthony J; Hooper, Nigel M

2007-01-01

Background Mammalian angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene. PMID:17597519

Effects of long-term heat stress and dietary restriction on the expression of genes of steroidogenic pathway and small heat-shock proteins in rat testicular tissue.

PubMed

Bozkaya, F; Atli, M O; Guzeloglu, A; Kayis, S A; Yildirim, M E; Kurar, E; Yilmaz, R; Aydilek, N

2017-08-01

The aim was to investigate the effects of long-term heat stress and dietary restriction on the expression of certain genes involving in steroidogenic pathway and small heat-shock proteins (sHSPs) in rat testis. Sprague Dawley rats (n = 24) were equally divided into four groups. Group I and II were kept at an ambient temperature of 22°C, while Groups III and IV were reared at 38°C for 9 weeks. Feed was freely available for Group I and Group III, while Group II and Group IV were fed 60% of the diet consumed by their ad libitum counterparts. At the end of 9 weeks, testicles were collected under euthanasia. Total RNA was isolated from testis tissue samples. Expression profiles of the genes encoding androgen-binding protein, follicle-stimulating hormone receptor, androgen receptor, luteinising hormone receptor, steroidogenic acute regulatory protein (StAR), cyclooxygenase-2 and sHSP genes were assessed at mRNA levels using qPCR. Long-term heat stress decreased the expression of StAR and HspB10 genes while dietary restriction upregulated StAR gene expression. The results suggested that long-term heat stress negatively affected the expression of StAR and HspB10 genes and the dietary restriction was able to reverse negative effect of heat stress on the expression of StAR gene in rat testis. © 2016 Blackwell Verlag GmbH.

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

PubMed Central

Koch, K S; Gleiberman, A S; Aoki, T; Leffert, H L; Feren, A; Jones, A L; Fodor, E J

1995-01-01

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed. Images PMID:7739889

Genome-Wide Transcriptional Profiling of Skin and Dorsal Root Ganglia after Ultraviolet-B-Induced Inflammation

PubMed Central

Paterson, Kathryn J.; Sisignano, Marco; Schmid, Ramona; Rust, Werner; Hildebrandt, Tobias; Geisslinger, Gerd; Orengo, Christine; Bennett, David L.; McMahon, Stephen B.

2014-01-01

Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain. PMID:24732968

Primary structure, expression and chromosomal locus of a human homolog of rat ERK3.

PubMed

Meloche, S; Beatty, B G; Pellerin, J

1996-10-03

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

[Molecular cloning, expression of rat Msx-1 and Msx-2 during early embryo genesis and roles for mandibular chondrogenesis].

PubMed

Ishiguro, S

1999-03-01

Quail-chick chimera experiments have shown a contribution of carnial neural crest cells to the craniofacial skeletal elements. Moreover, tissue interactions between epithelial-mesenchymal interaction during early facial process development are required for both skeletal differentiation and morphogenesis. In this study, it was observed that Msx homeobox containing genes expressed in the facial process were important molecules of cartilage morphogenesis. Rat cDNAs were isolated and encoded by Msx-1 and -2, and then the expression patterns using in situ hybridization were investigated during early rat face development. These genes were correlatively expressed in the cranial neural crest forming area (E 9.5 dpc) and the facial process (E 12.5 dpc). Antisence inhibition of Msx genes in the E 12.5 mandibular process exhibited the alteration of their gene expression and cartilage patterns. Antisence inhibition of Msx-1 induced lack of the medial portion of cartilage, and antisence inhibition of Msx-2 enhanced chondrogenesis of mandibular process under the organ culture condition. Thus it was concluded that expression of Msx genes during mandibular process development comprises important signals of chondrogenesis.

Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aho, Hanne; Schwemmer, M.; Tessmann, D.

1996-03-01

The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon-intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5{prime}- and 3{prime}-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in themore » N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5{prime}-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. 30 refs., 9 figs.« less

Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor

PubMed Central

Liedtke, Wolfgang; Choe, Yong; Martí-Renom, Marc A.; Bell, Andrea M.; Denis, Charlotte S.; Šali, Andrej; Hudspeth, A. J.; Friedman, Jeffrey M.; Heller, Stefan

2008-01-01

SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

Inflammatory factors mediate vulnerability to a social stress-induced depressive-like phenotype in passive coping rats

PubMed Central

Wood, Susan K.; Wood, Christopher S.; Lombard, Calliandra M.; Lee, Catherine S.; Zhang, Xiao-Yan; Finnell, Julie E.; Valentino, Rita J.

2014-01-01

Background Coping strategy impacts susceptibility to psychosocial stress. The locus coeruleus (LC) and dorsal raphe (DR) are monoamine nuclei that are implicated in stress-related disorders. This study was designed to identify genes in these nuclei that distinguish active and passive coping strategies in response to social stress. Methods Rats were exposed to repeated resident-intruder stress and coping strategy determined. Gene and protein expression in the LC and DR were determined by PCR array, ELISA, and compared between active and passive stress coping and unstressed rats. The effect of daily IL-1 receptor antagonist (IL-1ra, ICV) prior to stress on anhedonia was also determined. Results Rats exhibited passive or active coping strategies based on a short (SL) or longer latency (LL) to assume a defeat posture, respectively. Stress differentially regulated 19 and 26 genes in the LC and DR of SL and LL rats, respectively, many of which encoded for inflammatory factors. Notably, IL1β was increased in SL and decreased in LL rats in both the LC and DR. Protein changes were generally consistent with a proinflammatory response to stress in SL rats selectively. Stress produced anhedonia selectively in SL rats and this was prevented by IL-1ra, consistent with a role for IL1β in stress vulnerability. Conclusions This study highlighted distinctions in gene expression related to coping strategy in response to social stress. Passive coping was associated with a bias towards pro-inflammatory processes, particularly IL1β, whereas active coping and resistance to stress-related pathology was associated with suppression of inflammatory processes. PMID:25676490

Expression of the gene encoding the ghrelin receptor in rats selected for differential alcohol preference.

PubMed

Landgren, Sara; Engel, Jörgen A; Hyytiä, Petri; Zetterberg, Henrik; Blennow, Kaj; Jerlhag, Elisabet

2011-08-01

The mechanisms involved in alcohol use disorder, a chronic relapsing brain disorder, are complex and involve various signalling systems in the brain. Recently, the orexigenic peptide ghrelin was shown to be required for alcohol-induced reward, an effect mediated via ghrelin receptors, GHS-R1A, at the level of the cholinergic-dopaminergic reward link. Moreover, ghrelin increases and GHR-R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS-R1A gene has been associated with high alcohol consumption in humans. Therefore, GHS-R1A gene expression and alcohol intake were investigated in high, AA (Alko, Alcohol), versus low, ANA (Alko, Non-Alcohol), alcohol consuming rats as well as in Wistar rats. In the AA and ANA rats plasma ghrelin levels were also measured. GHS-R1A gene expression was increased in AA compared to ANA rats in nucleus accumbens, ventral tegmental area, amygdala, prefrontal cortex and hippocampus. A similar trend was observed in the ventral tegmental area of Wistar rats consuming high amounts of alcohol. Furthermore, the AA rats had significantly smaller reduction of plasma ghrelin levels over time, after several weeks of alcohol exposure, than had the ANA rats. The present study provides further evidence for that the ghrelin signalling system, in particular at the level of the mesocortocolimbic dopamine system, is involved in alcohol consumption, and thus possibly contributes to alcohol use disorder. Therefore the GHS-R1A may constitute a novel candidate for development of new treatment strategies for alcohol dependence. Copyright © 2011 Elsevier B.V. All rights reserved.

No involvement of the nerve growth factor gene locus in hypertension in spontaneously hypertensive rats.

PubMed

Nemoto, Kiyomitsu; Sekimoto, Masashi; Fukamachi, Katsumi; Kageyama, Haruaki; Degawa, Masakuni; Hamadai, Masanori; Hendley, Edith D; Macrae, I Mhairi; Clark, James S; Dominiczak, Anna F; Ueyama, Takashi

2005-02-01

Sympathetic hyper-innervation and increased levels of nerve growth factor (NGF), an essential neurotrophic factor for sympathetic neurons, have been observed in the vascular tissues of spontaneously hypertensive rats (SHRs). Such observations have suggested that the pathogenesis of hypertension might involve a qualitative or quantitative abnormality in the NGF protein, resulting from a significant mutation in the gene's promoter or coding region. In the present study, we analyzed the nucleotide sequences of the cis-element of the NGF gene in SHRs, stroke-prone SHRs (SHRSPs), and normotensive Wistar-Kyoto (WKY) rats. The present analyses revealed some differences in the 3-kb promoter region, coding exon, and 3' untranslated region (3'UTR) for the NGF gene among those strains. However, the observed differences did not lead to changes in promoter activity or to amino acid substitution; nor did they represent a link between the 3'UTR mutation of SHRSPs and elevated blood pressure in an F2 generation produced by crossbreeding SHRSPs with WKY rats. These results suggest that the NGF gene locus is not involved in hypertension in SHR/ SHRSP strains. The present study also revealed two differences between SHRs and WKY rats, as found in cultured vascular smooth muscle cells and in mRNA prepared from each strain. First, SHRs had higher expression levels of c-fos and c-jun genes, which encode the component of the AP-1 transcription factor that activates NGF gene transcription. Second, NGF mRNAs prepared from SHRs had a longer 3'UTR than those prepared from WKY rats. Although it remains to be determined whether these events play a role in the hypertension of SHR/SHRSP strains, the present results emphasize the importance of actively searching for aberrant trans-acting factor(s) leading to the enhanced expression of the NGF gene and NGF protein in SHR/SHRSP strains.

Lack of Humoral Immune Response to the Tetracycline (Tet) Activator in Rats Injected Intracranially with Tet-off rAAV Vectors

PubMed Central

Han, Ye; Chang, Qin A.; Virag, Tamas; West, Neva C.; George, David; Castro, Maria G.; Bohn, Martha C.

2010-01-01

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression. PMID:20164859

The Streptococcus mutans Serine/Threonine Kinase, PknB, Regulates Competence Development, Bacteriocin Production, and Cell Wall Metabolism ▿

PubMed Central

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R.

2010-01-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB. PMID:20231406

The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism.

PubMed

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R

2010-05-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.

Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene.

PubMed

Yamada, Toshiyuki; Nanashima, Naoki; Shimizu, Takeshi; Nakazawa, Yosuke; Nakazawa, Mitsuru; Tsuchida, Shigeki

2015-10-15

From our stock of SDRs (Sprague-Dawley rats), we established a mutant strain having small opaque eyes and named it HiSER (Hirosaki small-eye rat). The HiSER phenotype is progressive and autosomal recessive. In HiSER eyes, disruption and involution of the lens, thickening of the inner nuclear layer, detachment and aggregation of the retina, rudimentary muscle in the ciliary body and cell infiltration in the vitreous humour were observed. Genetic linkage analysis using crossing with Brown Norway rat suggested that the causative gene(s) is located on chromosome 10. Microarray analysis showed that the expression level of the Cryba1 gene encoding βA3/A1-crystallin on chromosome 10 was markedly decreased in HiSER eyes. Genomic PCR revealed deletion of a 3.6-kb DNA region encompassing exons 4-6 of the gene in HiSERs. In HiSER eyes, a chimaeric transcript of the gene containing exons 1-3 and an approximately 250-bp sequence originating from the 3'-UTR of the Nufip2 gene, located downstream of the breakpoint in the opposite direction, was present. Whereas the chimaeric transcript was expressed in HiSER eyes, neither normal nor chimaeric βA3/A1-crystallin proteins were detected by Western blot analysis. Real-time RT (reverse transcription)-PCR analysis revealed that expression level of the Nufip2 gene in the HiSER eye was 40% of that in the SDR eye. These results suggest that the disappearance of the βA3/A1-crystallin protein and, in addition, down-regulation of the Nufip2 gene as a consequence of gene rearrangement causes the HiSER phenotype. © 2015 Authors; published by Portland Press Limited.

Conjugated linoleic acid-enriched butter improved memory and up-regulated phospholipase A2 encoding-genes in rat brain tissue.

PubMed

Gama, Marco A S; Raposo, Nádia R B; Mury, Fábio B; Lopes, Fernando C F; Dias-Neto, Emmanuel; Talib, Leda L; Gattaz, Wagner F

2015-10-01

Reduced phospholipase A2 (PLA2) activity has been reported in blood cells and in postmortem brains of patients with Alzheimer disease (AD), and there is evidence that conjugated linoleic acid (CLA) modulates the activity of PLA2 groups in non-brain tissues. As CLA isomers were shown to be actively incorporated and metabolized in the brains of rats, we hypothesized that feeding a diet naturally enriched in CLA would affect the activity and expression of Pla 2 -encoding genes in rat brain tissue, with possible implications for memory. To test this hypothesis, Wistar rats were trained for the inhibitory avoidance task and fed a commercial diet (control) or experimental diets containing either low CLA- or CLA-enriched butter for 4 weeks. After this period, the rats were tested for memory retrieval and killed for tissue collection. Hippocampal expression of 19 Pla 2 genes was evaluated by qPCR, and activities of PLA2 groups (cPLA2, iPLA2, and sPLA2) were determined by radioenzymatic assay. Rats fed the high CLA diet had increased hippocampal mRNA levels for specific PLA2 isoforms (iPla 2 g6γ; cPla 2 g4a, sPla 2 g3, sPla 2 g1b, and sPla 2 g12a) and higher enzymatic activity of all PLA2 groups as compared to those fed the control and the low CLA diet. The increment in PLA2 activities correlated significantly with memory enhancement, as assessed by increased latency in the step-down inhibitory avoidance task after 4 weeks of treatment (rs = 0.69 for iPLA2, P < 0.001; rs = 0.81 for cPLA2, P < 0.001; and rs = 0.69 for sPLA2, P < 0.001). In face of the previous reports showing reduced PLA2 activity in AD brains, the present findings suggest that dairy products enriched in cis-9, trans-11 CLA may be useful in the treatment of this disease.

Intranasal gene delivery for treating Parkinson's disease: overcoming the blood-brain barrier.

PubMed

Aly, Amirah E-E; Waszczak, Barbara L

2015-01-01

Developing a disease-modifying gene therapy for Parkinson's disease (PD) has been a high priority for over a decade. However, due to the inability of large biomolecules to cross the blood-brain barrier (BBB), the only means of delivery to the brain has been intracerebral infusion. Intranasal administration offers a non-surgical means of bypassing the BBB to deliver neurotrophic factors, and the genes encoding them, directly to the brain. This review summarizes: i) evidence demonstrating intranasal delivery to the brain of a number of biomolecules having therapeutic potential for various CNS disorders; and ii) evidence demonstrating neuroprotective efficacy of a subset of biomolecules specifically for PD. The intersection of these two spheres represents the area of opportunity for development of new intranasal gene therapies for PD. To that end, our laboratory showed that intranasal administration of glial cell line-derived neurotrophic factor (GDNF), or plasmid DNA nanoparticles encoding GDNF, provides neuroprotection in a rat model of PD, and that the cells transfected by the nanoparticle vector are likely to be pericytes. A number of genes encoding neurotrophic factors have therapeutic potential for PD, but few have been tested by the intranasal route and shown to be neuroprotective in a model of PD. Intranasal delivery provides a largely unexplored, promising approach for development of a non-invasive gene therapy for PD.

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

Sex Differences in Behavior and Expression of CGRP-related Genes in a Rodent Model of Chronic Migraine

PubMed Central

Stucky, Nicholas L.; Gregory, Eugene; Winter, Michelle K.; He, Yong-Yue; Hamilton, Eric S.; McCarson, Kenneth E.; Berman, Nancy E.J.

2014-01-01

a. Objective The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of CGRP and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. b. Background Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (PBS) to the dura were measured to model changes in routine activity and allodynia. Calcitonin gene related peptide (CGRP), an important mediator of migraine pathogenesis, and the three components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. c. Methods Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 microliter volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 min post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. d. Results Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µl IS groups), and males showed increased perimasseter withdrawal responses (20 µl IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP related genes were upregulated by both IS and PBS applications. e. Conclusions This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation. PMID:21521205

Long-term erythropoietin gene expression from transduced cells in bioisolator devices.

PubMed

Yanay, Ofer; Barry, Simon C; Flint, Lisa Y; Brzezinski, Margaret; Barton, Randall W; Osborne, William R A

2003-11-20

Recombinant erythropoietin (EPO) is widely administered for long-term treatment of anemia associated with renal failure and other chronic diseases. The ability to deliver EPO by gene therapy would have clinical and economic benefit. We compared autologous and allogeneic transduced primary vascular smooth muscle cells for their ability to provide sustained EPO gene expression when encapsulated in TheraCyte devices implanted subcutaneously (SQ) or intraperitoneally (IP) in rats. Cells were transduced with retrovirus vector LrEpSN encoding rat EPO cDNA. Rats that received either autologous or allogeneic transduced cells showed elevated hematocrits (HCTs) ranging from 50 to 79% that were sustained for more than 12 months. The HCT of control rats remained at baseline (45.8%). Rats that received second SQ implants of either autologous or allogeneic cells showed elevations in hematocrit that were sustained for up to 12 months, suggesting the absence of immunological responses to transduced cells or implant material. All experimental groups had statistically significant elevated HCT (p < 0.001) when compared with controls. Both SQ and IP implantation were equally effective in delivering EPO long term. There were no significant differences in white blood cell (WBC) or platelet (PLT) values between treated and control animals. Implantation of TheraCyte devices was well tolerated and histological evaluation of the devices up to 12 months after surgery revealed a high degree of vascularization and no evidence of host immune response. TheraCyte devices offer a simple and safe gene delivery system that provides sustained therapeutic gene expression, permit removal and implantation of new devices, and do not require immunosuppression of the host.

Odors regulate Arc expression in neuronal ensembles engaged in odor processing.

PubMed

Guthrie, K; Rayhanabad, J; Kuhl, D; Gall, C

2000-06-26

Synaptic activity is critical to developmental and plastic processes that produce long-term changes in neuronal connectivity and function. Genes expressed by neurons in an activity-dependent fashion are of particular interest since the proteins they encode may mediate neuronal plasticity. One such gene encodes the activity-regulated cytoskeleton-associated protein, Arc. The present study evaluated the effects of odor stimulation on Arc expression in rat olfactory bulb. Arc mRNA was rapidly increased in functionally linked cohorts of neurons topographically activated by odor stimuli. These included neurons surrounding individual glomeruli, mitral cells and transynaptically activated granule cells. Dendritic Arc immunoreactivity was also increased in odor-activated glomeruli. Our results suggest that odor regulation of Arc expression may contribute to activity-dependent structural changes associated with olfactory experience.

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feder, J.N.; Jan, L.Y.; Jan, Y.N.

The Drosophila hairy gene encodes a basic helix- loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, the authors report the genomic cloning of the human hairy gene homolog (HRY). The coding region of themore » gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5[prime] to the coding region reveals a putatitve untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, they sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization. 34 refs., 4 figs., 1 tab.« less

The left end of rat L1 (L1Rn, long interspersed repeated) DNA which is a CpG island can function as a promoter.

PubMed Central

Nur, I; Pascale, E; Furano, A V

1988-01-01

Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line. Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter. The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis. Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts. The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed. Images PMID:2459662

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

PubMed

Ericsson, A; Geenen, V; Robert, F; Legros, J J; Vrindts-Gevaert, Y; Franchimont, P; Brene, S; Persson, H

1990-08-01

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.

Circadian rhythm of the Leydig cells endocrine function is attenuated during aging.

PubMed

Baburski, Aleksandar Z; Sokanovic, Srdjan J; Bjelic, Maja M; Radovic, Sava M; Andric, Silvana A; Kostic, Tatjana S

2016-01-01

Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups. Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor (Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-month group. The majority of examined clock genes displayed circadian behavior in expression but only a few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore, aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell's endocrine function decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance during aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes.

PubMed

Marcondes, Rodrigo Rodrigues; Carvalho, Kátia Cândido; Giannocco, Gisele; Duarte, Daniele Coelho; Garcia, Natália; Soares-Junior, José Maria; da Silva, Ismael Dale Cotrim Guerreiro; Maliqueo, Manuel; Baracat, Edmund Chada; Maciel, Gustavo Arantes Rosa

2017-08-01

Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-β and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Induced expression of hepatic N-methyl-D-aspartate receptor 2C subunit gene during liver enlargement induced by lead nitrate, a hepatocellular mitogen.

PubMed

Nemoto, Kiyomitsu; Ikeda, Ayaka; Hikida, Tokihiro; Kojima, Misaki; Degawa, Masakuni

2013-02-01

We previously demonstrated the super-induced expression of the Grin2c gene encoding the N-methyl-D-aspartate receptor 2C subunit during the development of liver enlargement with hepatocellular hypertrophy induced by phenobarbital, clofibrate, or piperonyl butoxide. In the present study, we assessed whether or not Grin2c gene expression was induced during the development of chemically induced liver enlargement with hyperplasia. Male Sprague-Dawley (SD) rats, stroke-prone spontaneously hypertensive rats (SHRSPs), and SHRSP's normotensive control, Wistar-Kyoto (WKY) rats, were administered lead nitrate (LN) (0.1 mmol/kg, single i.v.), a direct inducer of liver hyperplasia, and changes in the level of Grin2c mRNA in the liver were assessed by real-time RT-PCR. The level of hepatic Grin2c mRNA was significantly higher 6-48 hr after the injection in SD rats (about 30~40- and 70-fold over the control at 6~24 hr and 48 hr, respectively) and in WKY rats (about 20-fold over the control only at 12 hr), but was not significantly higher in SHRSPs. Such differences in LN-induced levels of Grin2c mRNA among SD rats, WKY rats, and SHRSPs were closely correlated with those in the previously reported increase in liver weight 48 hr after LN administration. The present findings suggest that the increase in the level of hepatic Grin2c mRNA relates to development of chemically induced liver enlargement with hyperplasia.

Biosynthesis and processing of the somatostatin family of peptide hormones.

PubMed

Andrews, P C; Dixon, J E

1986-01-01

Understanding of the biosynthesis of the somatostatin family of peptide hormones has greatly increased in recent years. Isolation and sequencing of the rat somatostatin gene indicates that it contains a single intron located between the codons for Gn(-57) and Glu(-56) of pre-prosomatostatin. The gene contains three repetitive sequences, one at the 5' end of the gene and two of them 3' to the coding portion. Two of the sequences consist of alternating purine-pyrimidine bases and have been shown to adopt Z-DNA structures in vitro. The cDNA for rat somatostatin codes for a 116-residue peptide structurally similar to the anglerfish and catfish precursors to the 14-residue somatostatin (SST-14). In addition to SST-14, the catfish and the anglerfish both contain an additional pancreatic somatostatin, each derived from a different gene. The catfish contains a 22-residue somatostatin, which is O-glycosylated at Thr-5. The second somatostatin gene from anglerfish encodes a prosomatostatin that is processed to a 28-residue peptide. The mature peptide contains a hydroxylated lysine at position 23.

Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

PubMed Central

Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

2016-01-01

Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

Molecular cloning and expression of the CRISP family of proteins in the boar.

PubMed

Vadnais, Melissa L; Foster, Douglas N; Roberts, Kenneth P

2008-12-01

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

PubMed

Bachvarov, D R; Hess, J F; Menke, J G; Larrivée, J F; Marceau, F

1996-05-01

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.

A role for tachykinins in female mouse and rat reproductive function.

PubMed

Pintado, C Oscar; Pinto, Francisco M; Pennefather, Jocelyn N; Hidalgo, Agustin; Baamonde, Ana; Sanchez, Teresa; Candenas, M Luz

2003-09-01

Tachykinins may be involved in reproduction. A reverse transcription-polymerase chain reaction assay was used to analyze the expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. The preprotachykinin (PPT) genes, PPT-A, PPT-B and PPT-C, that encode substance P/neurokinin A, neurokinin B, and hemokinin-1, respectively, and the genes that encode the tachykinin NK1, NK2, and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of neprilysin (NEP), the main enzyme involved in tachykinin metabolism, was also expressed in the uterus. Isolated cumulus granulosa cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of the tachykinin NK1 and NK2 receptors. Mouse oocytes expressed PPT-A and -B mRNA transcripts. A low expression of the three tachykinin receptors was observed but PPT-C and NEP were undetectable. Two- and 8- to 16-cell mouse embryos expressed only a low-abundance transcript corresponding to the NK1 receptor. However, the mRNAs of PPT-B, PPT-C and NEP appeared in blastocyst-stage embryos. A low-abundance transcript corresponding to the NK2 receptor was the only target gene detected in mice sperm. Female mice or rats treated neonatally with capsaicin showed a reduced fertility. A reduction in litter size was observed in female rats treated in vivo with the tachykinin NK3 receptor antagonist SR 142801. These data show that tachykinins of both neuronal and nonneuronal origin are differentially expressed in various types of reproductive cells and may play a role in female reproductive function.

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

PubMed

Graw, J; Liebstein, A; Pietrowski, D; Schmitt-John, T; Werner, T

1993-12-22

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Gene transfer of high-mobility group box 1 box-A domain in a rat acute liver failure model.

PubMed

Tanaka, Masayuki; Shinoda, Masahiro; Takayanagi, Atsushi; Oshima, Go; Nishiyama, Ryo; Fukuda, Kazumasa; Yagi, Hiroshi; Hayashida, Tetsu; Masugi, Yohei; Suda, Koichi; Yamada, Shingo; Miyasho, Taku; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Obara, Hideaki; Itano, Osamu; Takeuchi, Hiroya; Sakamoto, Michiie; Tanabe, Minoru; Maruyama, Ikuro; Kitagawa, Yuko

2015-04-01

High-mobility group box 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. The protein encoded by the box-A domain of the HMGB1 gene is known to act as a competitive inhibitor of HMGB1. In this study, we investigated whether box-A gene transfer results in box-A protein production in rats and assessed therapeutic efficacy in vivo using an acute liver failure (ALF) model. Three types of adenovirus vectors were constructed-a wild type and two mutants-and a mutant vector was then selected based on the secretion from HeLa cells. The secreted protein was subjected to a tumor necrosis factor (TNF) production inhibition test in vitro. The vector was injected via the portal vein in healthy Wistar rats to confirm box-A protein production in the liver. The vector was then injected via the portal vein in rats with ALF. Western blot analysis showed enhanced expression of box-A protein in HeLa cells transfected with one of the mutant vectors. The culture supernatant from HeLa cells transfected with the vector inhibited TNF-α production from macrophages. Expression of box-A protein was confirmed in the transfected liver at 72 h after transfection. Transfected rats showed decreased hepatic enzymes, plasma HMGB1, and hepatic TNF-α messenger RNA levels, and histologic findings and survival were significantly improved. HMGB1 box-A gene transfer results in box-A protein production in the liver and appears to have a beneficial effect on ALF in rats. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat.

PubMed

Schuhmacher, Laura-Nadine; Smith, Ewan St John

2016-12-13

Acid-sensing ion channels (ASICs) are a family of ion channels comprised of six subunits encoded by four genes and they are expressed throughout the peripheral and central nervous systems. ASICs have been implicated in a wide range of physiological and pathophysiological processes: pain, breathing, synaptic plasticity and excitotoxicity. Unlike mice and humans, naked mole-rats do not perceive acid as a noxious stimulus, even though their sensory neurons express functional ASICs, likely an adaptation to living in a hypercapnic subterranean environment. Previous studies of ASIC expression in the mammalian nervous system have often not examined all subunits, or have failed to adequately quantify expression between tissues; to date there has been no attempt to determine ASIC expression in the central nervous system of the naked mole-rat. Here we perform a geNorm study to identify reliable housekeeping genes in both mouse and naked mole-rat and then use quantitative real-time PCR to estimate the relative amounts of ASIC transcripts in different tissues of both species. We identify RPL13A (ribosomal protein L13A) and CANX (calnexin), and β-ACTIN and EIF4A (eukaryotic initiation factor 4a) as being the most stably expressed housekeeping genes in mouse and naked mole-rat, respectively. In both species, ASIC3 was most highly expressed in dorsal root ganglia (DRG), and ASIC1a, ASIC2b and ASIC3 were more highly expressed across all brain regions compared to the other subunits. We also show that ASIC4, a proton-insensitive subunit of relatively unknown function, was highly expressed in all mouse tissues apart from DRG and hippocampus, but was by contrast the lowliest expressed ASIC in all naked mole-rat tissues.

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

PubMed

Figueiredo, B C; Almazan, G; Ma, Y; Tetzlaff, W; Miller, F D; Cuello, A C

1993-03-01

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat

PubMed Central

Boehn, Susanne N.E.; Spahn, Sonja; Neudecker, Sabine; Keppler, Andrea; Bihoreau, Marie-Thérèse; Kränzlin, Bettina; Pandey, Priyanka; Hoffmann, Sigrid C.; Li, Li; Torres, Vicente E.; Gröne, Hermann-Josef; Gretz, Norbert

2013-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. Methods Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. Results Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. Conclusions Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD. PMID:23543593

Behaviorally activated mRNA expression profiles produce signatures of learning and enhanced inhibition in aged rats with preserved memory.

PubMed

Haberman, Rebecca P; Colantuoni, Carlo; Koh, Ming Teng; Gallagher, Michela

2013-01-01

Aging is often associated with cognitive decline, but many elderly individuals maintain a high level of function throughout life. Here we studied outbred rats, which also exhibit individual differences across a spectrum of outcomes that includes both preserved and impaired spatial memory. Previous work in this model identified the CA3 subfield of the hippocampus as a region critically affected by age and integral to differing cognitive outcomes. Earlier microarray profiling revealed distinct gene expression profiles in the CA3 region, under basal conditions, for aged rats with intact memory and those with impairment. Because prominent age-related deficits within the CA3 occur during neural encoding of new information, here we used microarray analysis to gain a broad perspective of the aged CA3 transcriptome under activated conditions. Behaviorally-induced CA3 expression profiles differentiated aged rats with intact memory from those with impaired memory. In the activated profile, we observed substantial numbers of genes (greater than 1000) exhibiting increased expression in aged unimpaired rats relative to aged impaired, including many involved in synaptic plasticity and memory mechanisms. This unimpaired aged profile also overlapped significantly with a learning induced gene profile previously acquired in young adults. Alongside the increased transcripts common to both young learning and aged rats with preserved memory, many transcripts behaviorally-activated in the current study had previously been identified as repressed in the aged unimpaired phenotype in basal expression. A further distinct feature of the activated profile of aged rats with intact memory is the increased expression of an ensemble of genes involved in inhibitory synapse function, which could control the phenotype of neural hyperexcitability found in the CA3 region of aged impaired rats. These data support the conclusion that aged subjects with preserved memory recruit adaptive mechanisms to retain tight control over excitability under both basal and activated conditions.

Gene transcripts selectively down-regulated in the shell of the nucleus accumbens long after heroin self-administration are up-regulated in the core independent of response contingency.

PubMed

Jacobs, Edwin H; de Vries, Taco J; Smit, August B; Schoffelmeer, Anton N M

2004-01-01

Long-term drug-induced alterations in neurotransmission within the nucleus accumbens (NAc) shell and core may underlie relapse to drug-seeking behavior and drug-taking upon re-exposure to drugs and drug-associated stimuli (cues) during abstinence. Using an open screening strategy, we recently identified 25 gene transcripts, encoding for proteins involved in neuronal functioning and structure that are down-regulated in rat NAc shell after contingent (active), but not after non-contingent (passive), heroin administration. Studying the expression of the same transcripts in the NAc core by means of quantitative PCR, we now demonstrate that most of these transcripts are up-regulated in that NAc subregion long (3 weeks) after heroin self-administration in rats. A similar up-regulation in gene expression was also apparent in the NAc core of animals with a history of non-contingent heroin administration (yoked controls). These data indicate that heroin self-administration differentially regulates genes in the NAc core as compared with the shell. Moreover, whereas cognitive processes involved in active drug self-administration (e.g., instrumental learning) seems to direct gene expression in the NAc shell, neuroplasticity in the NAc core may be due to the pharmacological effects of heroin (including Pavlovian conditioning), as expressed in rats upon contingent as well as non-contingent administration of heroin.

Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, C.; Nettesheim, P.; Barrett, J.C.

1987-04-01

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injectedmore » into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approx. = 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.« less

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

PubMed Central

Williams, C M; Coleman, J W

1995-01-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs. Images Figure 1 Figure 2 Figure 3 PMID:7490125

Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions.

PubMed

Loh, Su-Yi; Jahans-Price, Thomas; Greenwood, Michael P; Greenwood, Mingkwan; Hoe, See-Ziau; Konopacka, Agnieszka; Campbell, Colin; Murphy, David; Hindmarch, Charles C T

2017-01-01

The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2 . Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON.

Comparative genomics on Norrie disease gene.

PubMed

Katoh, Masuko; Katoh, Masaru

2005-05-01

DAND1 (NBL1), DAND2 (CKTSF1B1 or GREM1 or GREMLIN), DAND3 (CKTSF1B2 or GREM2 or PRDC), DAND4 (CER1), DAND5 (CKTSF1B3 or GREM3 or DANTE), MUC2, MUC5AC, MUC5B, MUC6, MUC19, WISP1, WISP2, WISP3, VWF, NOV and Norrie disease (NDP or NORRIN) genes encode proteins with cysteine knot domain. Cysteine-knot superfamily proteins regulate ligand-receptor interactions for a variety of signaling pathways implicated in embryogenesis, homeostasis, and carcinogenesis. Although Ndp is unrelated to Wnt family members, Ndp is claimed to function as a ligand for Fzd4. Here, we identified and characterized rat Ndp, cow Ndp, chicken ndp and zebrafish ndp genes by using bioinformatics. Rat Ndp gene, consisting of three exons, was located within AC105563.4 genome sequence. Cow Ndp and chicken ndp complete CDS were derived from CB467544.1 EST and BX932859.2 cDNA, respectively. Zebrafish ndp gene was located within BX572627.5 genome sequence. Rat Ndp (131 aa) was a secreted protein with C-terminal cysteine knot-like (CTCK) domain. Rat Ndp showed 100, 96.9, 95.4, 87.8 and 66.4 total-amino-acid identity with mouse Ndp, cow Ndp, human NDP, chicken ndp and zebrafish ndp, respectively. Exon-intron structure of mammalian Ndp orthologs was well conserved. FOXA2, CUTL1 (CCAAT displacement protein), LMO2, CEBPA (C/EBPalpha)-binding sites and triple POU2F1 (OCT1)-binding sites were conserved among promoters of mammalian Ndp orthologs.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

PubMed

Tschernutter, M; Schlichtenbrede, F C; Howe, S; Balaggan, K S; Munro, P M; Bainbridge, J W B; Thrasher, A J; Smith, A J; Ali, R R

2005-04-01

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puntel, Mariana; Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048

2013-05-01

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, andmore » can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at acute and chronic time points. ► Doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered in the brain. ► Efficacy and safety of HC-Ad-TK/TetOn-Flt3L merits its use in a GBM Phase I trial.« less

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

Profiling of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR).

PubMed

Boylan, Joan M; Sanders, Jennifer A; Neretti, Nicola; Gruppuso, Philip A

2015-07-01

The mechanistic target of rapamycin (mTOR) integrates growth factor signaling, nutrient abundance, cell growth, and proliferation. On the basis of our interest in somatic growth in the late gestation fetus, we characterized the role of mTOR in the regulation of hepatic gene expression and translation initiation in fetal and adult rats. Our strategy was to manipulate mTOR signaling in vivo and then characterize the transcriptome and translating mRNA in liver tissue. In adult rats, we used the nonproliferative growth model of refeeding after a period of fasting and the proliferative model of liver regeneration following partial hepatectomy. We also studied livers from preterm fetal rats (embryonic day 19) in which fetal hepatocytes are asynchronously proliferating. All three models employed rapamycin to inhibit mTOR signaling. Analysis of the transcriptome in fasted-refed animals showed rapamycin-mediated induction of genes associated with oxidative phosphorylation. Genes associated with RNA processing were downregulated. In liver regeneration, rapamycin induced genes associated with lysosomal metabolism, steroid metabolism, and the acute phase response. In fetal animals, rapamycin inhibited expression of genes in several functional categories that were unrelated to effects in the adult animals. Translation control showed marked fetal-adult differences. In both adult models, rapamycin inhibited the translation of genes with complex 5' untranslated regions, including those encoding ribosomal proteins. Fetal translation was resistant to the effects of rapamycin. We conclude that the mTOR pathway in liver serves distinct physiological roles in the adult and fetus, with the latter representing a condition of rapamycin resistance. Copyright © 2015 the American Physiological Society.

Evidence of Neurobiological Changes in the Presymptomatic PINK1 Knockout Rat.

PubMed

Ferris, Craig F; Morrison, Thomas R; Iriah, Sade; Malmberg, Samantha; Kulkarni, Praveen; Hartner, Jochen C; Trivedi, Malav

2018-01-01

Genetic models of Parkinson's disease (PD) coupled with advanced imaging techniques can elucidate neurobiological disease progression, and can help identify early biomarkers before clinical signs emerge. PTEN-induced putative kinase 1 (PINK1) helps protect neurons from mitochondrial dysfunction, and a mutation in the associated gene is a risk factor for recessive familial PD. The PINK1 knockout (KO) rat is a novel model for familial PD that has not been neuroradiologically characterized for alterations in brain structure/function, alongside behavior, prior to 4 months of age. To identify biomarkers of presymptomatic PD in the PINK1 -/- rat at 3 months using magnetic resonance imaging techniques. At postnatal weeks 12-13; one month earlier than previously reported signs of motor and cognitive dysfunction, this study combined imaging modalities, including assessment of quantitative anisotropy across 171 individual brain areas using an annotated MRI rat brain atlas to identify sites of gray matter alteration between wild-type and PINK1 -/- rats. The olfactory system, hypothalamus, thalamus, nucleus accumbens, and cerebellum showed differences in anisotropy between experimental groups. Molecular analyses revealed reduced levels of glutathione, ATP, and elevated oxidative stress in the substantia nigra, striatum and deep cerebellar nuclei. Mitochondrial genes encoding proteins in Complex IV, along with mRNA levels associated with mitochondrial function and genes involved in glutathione synthesis were reduced. Differences in brain structure did not align with any cognitive or motor impairment. These data reveal early markers, and highlight novel brain regions involved in the pathology of PD in the PINK1 -/- rat before behavioral dysfunction occurs.

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H.

PubMed

Lynes, John; Wibowo, Mia; Koschmann, Carl; Baker, Gregory J; Saxena, Vandana; Muhammad, A K M G; Bondale, Niyati; Klein, Julia; Assi, Hikmat; Lieberman, Andrew P; Castro, Maria G; Lowenstein, Pedro R

2014-07-01

In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.

A subchronic feeding study of dicamba-tolerant soybean with the dmo gene in Sprague-Dawley rats.

PubMed

Wang, Xiaoyun; He, Xiaoyun; Zou, Shiyin; Xu, Wentao; Jia, Xin; Zhao, Bo; Zhao, Changhui; Huang, Kunlun; Liang, Zhihong

2016-06-01

The dicamba-tolerant soybean MON87708 expresses the dicamba mono-oxygenase (DMO) enzyme that is encoded by the dmo gene. In order to evaluate the safety of this soybean, a 90-day subchronic feeding toxicity study (13 weeks) was conducted on Sprague-Dawley rats. A total of 140 rats were divided into 7 groups (10/sex/group), including a standard commercial diet control group. The genetically modified (GM) soybean MON87708 and the near isogenic non-GM soybean A3525 were respectively processed to unhulled, full-fat, and heat-treated powder, then mixed into the diet at levels of 7.5%, 15%, and 30% (wt/wt) with the main nutrients of the various diets balanced and then fed to 6 groups. The remaining group of rats fed with a commercial rat diet served as blank control. Some isolated parameters indicated statistically significant differences in body weight, feed consumption/utilization, hematology, serum biochemistry, and relative organ weights. These differences were not consistent across gender or test-diet dose, which were attributed to incidental and biological variability. In conclusion, the results demonstrated that the transgenic soybean MON87708 containing DMO was as safe as non-transgenic isogenic counterpart with historical safe use. Copyright © 2016 Elsevier Inc. All rights reserved.

[Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis].

PubMed

Zhang, Xiao-dong; Gou, Da-wei; Miao, Shi-ying; Zhang, Jian-chao; Zong, Shu-dong; Wang, Lin-fang

2003-06-01

To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. (1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells. Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

PubMed

Touchard, E; Heiduschka, P; Berdugo, M; Kowalczuk, L; Bigey, P; Chahory, S; Gandolphe, C; Jeanny, J-C; Behar-Cohen, F

2012-09-01

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 μg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

PubMed

Davies, Kelvin P; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C; Desai, Pratik; Verselis, Vytas K; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J

2007-10-01

Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif.

PubMed

Chan, Y L; Paz, V; Olvera, J; Wool, I G

1993-04-30

The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939. Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene. The mRNA for the protein is about 500 nucleotides in length. Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37. We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37.

Locus ceruleus control of state-dependent gene expression.

PubMed

Cirelli, Chiara; Tononi, Giulio

2004-06-09

Wakefulness and sleep are accompanied by changes in behavior and neural activity, as well as by the upregulation of different functional categories of genes. However, the mechanisms responsible for such state-dependent changes in gene expression are unknown. Here we investigate to what extent state-dependent changes in gene expression depend on the central noradrenergic (NA) system, which is active in wakefulness and reduces its firing during sleep. We measured the levels of approximately 5000 transcripts expressed in the cerebral cortex of control rats and in rats pretreated with DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], a neurotoxin that removes the noradrenergic innervation of the cortex. We found that NA depletion reduces the expression of approximately 20% of known wakefulness-related transcripts. Most of these transcripts are involved in synaptic plasticity and in the cellular response to stress. In contrast, NA depletion increased the expression of the sleep-related gene encoding the translation elongation factor 2. These results indicate that the activity of the central NA system during wakefulness modulates neuronal transcription to favor synaptic potentiation and counteract cellular stress, whereas its inactivity during sleep may play a permissive role to enhance brain protein synthesis.

THE INVOLVEMENT OF HUMAN MONOGENIC CARDIOMYOPATHY GENES IN EXPERIMENTAL POLYGENIC CARDIAC HYPERTROPHY.

PubMed

Prestes, Priscilla R; Marques, Francine Z; Lopez-Campos, Guillermo; Lewandowski, Paul; Delbridge, Lea M D; Charchar, Fadi J; Harrap, Stephen B

2018-05-18

Hypertrophic cardiomyopathy thickens heart muscles reducing functionality and increasing risk of cardiac disease and morbidity. Genetic factors are involved, but their contribution is poorly understood. We used the hypertrophic heart rat (HHR), a unique normotensive polygenic model of cardiac hypertrophy and heart failure to investigate the role of genes associated with monogenic human cardiomyopathy. We selected 42 genes involved in monogenic human cardiomyopathies to study: 1) DNA variants, by sequencing the whole-genome of 13-week old HHR and age-matched normal heart rat (NHR), its genetic control strain; 2) mRNA expression, by targeted RNA-sequencing in left ventricles of HHR and NHR at five ages (2-days old, 4-, 13-, 33- and 50-weeks old) compared to human idiopathic dilated data; and 3) microRNA expression, with rat microRNA microarrays in left ventricles of 2-days old HHR and age-matched NHR. We also investigated experimentally validated microRNA-mRNA interactions. Whole-genome sequencing revealed unique variants mostly located in non-coding regions of HHR and NHR. We found 29 genes differentially expressed in at least one age. Genes encoding desmoglein 2 (Dsg2) and transthyretin (Ttr) were significantly differentially expressed at all ages in the HHR, but only Ttr was also differentially expressed in human idiopathic cardiomyopathy. Lastly, only two microRNAs differentially expressed in the HHR were present in our comparison of validated microRNA-mRNA interactions. These two microRNAs interact with five of the genes studied. Our study shows that genes involved in monogenic forms of human cardiomyopathies may also influence polygenic forms of the disease.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1.

PubMed

Chin, H; Krall, M; Kim, H L; Kozak, C A; Mock, B

1992-12-01

Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma

PubMed Central

2011-01-01

Background Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer. PMID:21232125

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

PubMed

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.

PubMed

Konopiński, R; Nowak, R; Siedlecki, J A

1996-10-17

Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.

PubMed

Atta, Hussein; El-Rehany, Mahmoud; Hammam, Olfat; Abdel-Ghany, Hend; Ramzy, Maggie; Roderfeld, Martin; Roeb, Elke; Al-Hendy, Ayman; Raheim, Salama Abdel; Allam, Hatem; Marey, Heba

2014-01-01

Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin

PubMed Central

Yadav, Anamika; Mahimainathan, Lenin; Valente, Anthony J.

2011-01-01

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1 (encodes hamartin) and TSC-2 (encodes tuberin). In humans, deficiency in TSC1/2 is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSC function in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTEN was highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser2448 of mTOR and Thr389 of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53 as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53 may prevent progression of kidney cancer in TSC patients. PMID:22737271

The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

PubMed

Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

1996-07-01

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

HMGB1 regulates P-glycoprotein expression in status epilepticus rat brains via the RAGE/NF-κB signaling pathway

PubMed Central

Xie, Yuan; Yu, Nian; Chen, Yan; Zhang, Kang; Ma, Hai-Yan; Di, Qing

2017-01-01

Overexpression of P-glycoprotein (P-gp) in the brain is an important mechanism involved in drug-resistant epilepsy (DRE). High-mobility group box 1 (HMGB1), an inflammatory cytokine, significantly increases following seizures and may be involved in upregulation of P-gp. However, the underlying mechanisms remain elusive. The aim of the present study was to evaluate the role of HMGB1 and its downstream signaling components, receptor for advanced glycation end-product (RAGE) and nuclear factor-κB (NF-κB), on P-gp expression in rat brains during status epilepticus (SE). Small interfering RNA (siRNA) was administered to rats prior to induction of SE by pilocarpine, to block transcription of the genes encoding HMGB1 and RAGE, respectively. An inhibitor of NF-κB, pyrrolidinedithiocarbamic acid (PDTC), was utilized to inhibit activation of NF-κB. The expression levels of HMGB1, RAGE, phosphorylated-NF-κB p65 (p-p65) and P-gp were detected by western blotting. The relative mRNA expression levels of the genes encoding these proteins were measured using reverse transcription-quantitative polymerase chain reaction and the cellular localization of the proteins was determined by immunofluorescence. Pre-treatment with HMGB1 siRNA reduced the expression levels of RAGE, p-p65 and P-gp. PDTC reduced the expression levels of P-gp. These findings suggested that overexpression of P-gp during seizures may be regulated by HMGB1 via the RAGE/NF-κB signaling pathway, and may be a novel target for treating DRE. PMID:28627626

Tissue-specific regulation of medium-chain acyl-CoA dehydrogenase gene by thyroid hormones in the developing rat.

PubMed

Djouadi, F; Riveau, B; Merlet-Benichou, C; Bastin, J

1997-05-15

During development, gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear-encoded mitochondrial enzyme that catalyses the first step of medium-chain fatty acid beta-oxidation, is highly regulated in tissues in accordance with fatty acid utilization, but the factors involved in this regulation are largely unknown. To investigate a possible role of thyroid hormones, rat pups were made hypothyroid by the administration of propylthiouracyl to the mother from day 12 of gestation, and their kidneys, heart and liver were removed on postnatal day 16 to determine MCAD mRNA abundance, protein level and enzyme activity. Similar experiments were run in 3,3',5-tri-iodothyronine (T3)-replaced hypothyroid (1 microg of T3/100 g body weight from postnatal day 5 to 15) and euthyroid pups. Hypothyroidism led to an increase in MCAD mRNA abundance in kidney and a decrease in abundance in heart, but had no effect in liver. The protein levels and enzyme activity were lowered in hypothyroid heart and kidney, suggesting that hypothyroidism affects post-transcriptional steps of gene expression in the kidney. All the effects of hypothyroidism were completely reversed in both heart and kidney by T3 replacement. Injection of a single T3 dose into 16-day-old euthyroid rats also led to tissue-specific changes in mRNA abundance. Nuclear run-on assays performed from hypothyroid and hypothyroid plus T3 rats showed that T3 stimulates MCAD gene transcription in heart and represses it in the kidney. These results indicate that the postnatal rise in circulating T3 is essential to the developmental regulation of the MCAD gene in vivo.

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Ex vivo adenoviral gene transfer of constitutively activated STAT3 reduces post-transplant liver injury and promotes regeneration in a 20% rat partial liver transplant model.

PubMed

Huda, Kamrul A S M; Guo, Lei; Haga, Sanae; Murata, Hiroshi; Ogino, Tetsuya; Fukai, Moto; Yagi, Takahito; Iwagaki, Hiromi; Tanaka, Noriaki; Ozaki, Michitaka

2006-05-01

Signal transducer and activator of transcription-3 (STAT3) is one of the most important transcription factors for liver regeneration. This study was designed to examine the effects of constitutively activated STAT3 (STAT3-C) on post-transplant liver injury and regeneration in a rat 20% partial liver transplant (PLTx) model by ex vivo adenoviral gene transfer. Adenovirus encoding the STAT3-C gene was introduced intraportally into liver grafts and clamped for 30 min during cold preservation. After orthotopic PLTx, liver graft/body weights and serum biochemistry were monitored, and both a histological study and DNA binding assay were performed. STAT3-C protein expression and its binding to DNA in the liver graft were confirmed by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. This treatment modality promoted post-Tx liver regeneration effectively and rapidly. The serum levels of alanine aminotransferase/aspartate aminotransferase (AST/ALT) and bilirubin decreased in rats with STAT3-C. However, albumin (a marker of liver function) did not. Ex vivo gene transfer of STAT3-C to liver grafts reduced post-Tx injury and promoted liver regeneration. Thus, the activation of STAT3 in the liver graft may be a potentially effective clinical strategy for improving the outcome of small-for-size liver transplantation.

General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats.

PubMed

Bloch, C A; Thorne, G M; Ausubel, F M

1989-07-01

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.

Multigeneration reproductive and developmental toxicity study of bar gene inserted into genetically modified potato on rats.

PubMed

Rhee, Gyu Seek; Cho, Dae Hyun; Won, Yong Hyuck; Seok, Ji Hyun; Kim, Soon Sun; Kwack, Seung Jun; Lee, Rhee Da; Chae, Soo Yeong; Kim, Jae Woo; Lee, Byung Mu; Park, Kui Lea; Choi, Kwang Sik

2005-12-10

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive-developmental ability.

The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

PubMed Central

Wieczorek, D F; Smith, C W; Nadal-Ginard, B

1988-01-01

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing. Images PMID:3352602

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

Comparative genome and evolutionary analysis of naturally occurring Beilong virus in brown and black rats.

PubMed

Woo, Patrick C Y; Wong, Annette Y P; Wong, Beatrice H L; Lam, Carol S F; Fan, Rachel Y Y; Lau, Susanna K P; Yuen, Kwok-Yung

2016-11-01

Recently, we reported the presence of Beilong virus in spleen and kidney samples of brown rats and black rats, suggesting that these rodents could be natural reservoirs of Beilong virus. In this study, four genomes of Beilong virus from brown rats and black rats were sequenced. Similar to the Beilong virus genome sequenced from kidney mesangial cell line culture, those of J-virus from house mouse and Tailam virus from Sikkim rats, these four genomes from naturally occurring Beilong virus also contain the eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'). In these four genomes, the attachment glycoprotein encoded by the G gene consists of 1046 amino acids; but for the original Beilong virus genome sequenced from kidney mesangial cell line, the G CDS was predicted to be prematurely terminated at position 2205 (TGG→TAG), resulting in a 734-amino-acid truncated G protein. This phenomenon of a lack of nonsense mutation in naturally occurring Beilong viruses was confirmed by sequencing this region of 15 additional rodent samples. Phylogenetic analyses showed that the cell line and naturally occurring Beilong viruses were closely clustered, without separation into subgroups. In addition, these viruses were further clustered with J-virus and Tailam virus, with high bootstrap supports of >90%, forming a distinct group in Paramyxoviridae. Brown rats and black rats are natural reservoirs of Beilong virus. Our results also supports that the recently proposed genus, Jeilongvirus, should encompass Beilong virus, J-virus and Tailam virus as members. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrodynamics based transfection in normal and fibrotic rats

PubMed Central

Yeikilis, Rita; Gal, Shunit; Kopeiko, Natalia; Paizi, Melia; Pines, Mark; Braet, Filip; Spira, Gadi

2006-01-01

AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect. CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced. PMID:17036386

Herbal Formula HT048 Attenuates Diet-Induced Obesity by Improving Hepatic Lipid Metabolism and Insulin Resistance in Obese Rats.

PubMed

Lee, Yoon Hee; Jin, Bora; Lee, Sung Hyun; Song, MiKyung; Bae, HyeonHui; Min, Byung Jae; Park, Juyeon; Lee, Donghun; Kim, Hocheol

2016-10-25

It is well established that obesity causes a variety of chronic diseases such as cardiovascular diseases and diabetes. Despite the diligent scientific efforts to find effective ways to lower the level of obesity, the size of obese population grows continuously around the world. Here we present the results that show feeding diet containing HT048, a mixture of the extracts of Crataegus pinnatifida leaves and Citrus unshiu peel, two of the well-known traditional herbal medicines in Eastern Asia, decreases obesity in rats. We fed rats with five different diets for 10 weeks: chow diet (STD), high-fat diet (HFD), high-fat diet with 0.04% orlistat, a drug to treat obesity (HFD + Orlistat), high-fat diet with 0.2% HT048 ( w / w ; HFD + 0.2% HT048), and high-fat diet with 0.6% HT048 ( w / w ; HFD + 0.6% HT048). It was found that both body and total white adipose tissue weight of HT048 groups significantly decreased compared to those of the HFD group. Moreover, HT048 decreased serum insulin levels in HFD-fed obese rats. At the molecular level, HT048 supplementation downregulated genes involved in lipogenesis, gluconeogenesis, and adipogenesis, while the expression level of β-oxidation genes was increased. Supplementation-drug interactions are not likely as HFD and HT048-containing diet did not significantly induce genes encoding CYPs. Collectively, this study suggests that HT048 taken as dietary supplement helps to decrease obesity and insulin resistance in HFD-fed obese rats.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

PubMed

Mollereau, C; Simons, M J; Soularue, P; Liners, F; Vassart, G; Meunier, J C; Parmentier, M

1996-08-06

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions

PubMed Central

Jahans-Price, Thomas; Greenwood, Michael P.; Greenwood, Mingkwan; Hoe, See-Ziau; Konopacka, Agnieszka

2017-01-01

Abstract The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2. Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON. PMID:29279858

Effects of the antiestrogen fulvestrant (ICI 182,780) on gene expression of the rat efferent ductules.

PubMed

Yasuhara, Fabiana; Gomes, Gisele Renata Oliveira; Siu, Erica Rosanna; Suenaga, Cláudia Igushi; Maróstica, Elisabeth; Porto, Catarina Segreti; Lazari, Maria Fatima Magalhaes

2008-09-01

The efferent ductules express the highest amount of estrogen receptors ESR1 (ERalpha) and ESR2 (ERbeta) within the male reproductive tract. Treatment of rats with the antiestrogen fulvestrant (ICI 182,780) causes inhibition of fluid reabsorption in the efferent ductules, leading to seminiferous tubule atrophy and infertility. To provide a more comprehensive knowledge about the molecular targets for estrogen in the rat efferent ductules, we investigated the effects of ICI 182,780 treatment on gene expression using a microarray approach. Treatment with ICI 182,780 increased or reduced at least 2-fold the expression of 263 and 98 genes, respectively. Not surprisingly, several genes that encode ion channels and macromolecule transporters were affected. Interestingly, treatment with ICI 182,780 markedly altered the expression of genes related to extracellular matrix organization. Matrix metalloproteinase 7 (Mmp7), osteopontin (Spp1), and neuronal pentraxin 1 (Nptx1) were among the most altered genes in this category. Upregulation of Mmp7 and Spp1 and downregulation of Nptx1 were validated by Northern blot. Increase in Mmp7 expression was further confirmed by immunohistochemistry and probably accounted for the decrease in collagen content observed in the efferent ductules of ICI 182,780-treated animals. Downregulation of Nptx1 probably contributed to the extracellular matrix changes and decreased amyloid deposition in the efferent ductules of ICI 182,780-treated animals. Identification of new molecular targets for estrogen action may help elucidate the regulatory role of this hormone in the male reproductive tract.

Preliminary Characterization of a Leptin Receptor Knockout Rat Created by CRISPR/Cas9 System.

PubMed

Bao, Dan; Ma, Yuanwu; Zhang, Xu; Guan, Feifei; Chen, Wei; Gao, Kai; Qin, Chuan; Zhang, Lianfeng

2015-11-05

Leptin receptor, which is encoded by the diabetes (db) gene and is highly expressed in the choroid plexus, regulatesenergy homeostasis, the balance between food intake and energy expenditure, fertility and bone mass. Here, using CRISPR/Cas9 technology, we created the leptin receptor knockout rat. Homozygous leptin receptor null rats are characterized by obesity, hyperphagia, hyperglycemia, glucose intolerance, hyperinsulinemia and dyslipidemia. Due to long-term poor glycemic control, the leptin receptor knockout rats also develop some diabetic complications such as pancreatic, hepatic and renal lesions. In addition, the leptin receptor knockout rats show a significant decrease in bone volume and bone mineral density of the femur compared with their wild-type littermates. Our model has rescued some deficiency of the existing rodent models, such as the transient hyperglycemia of db/db mice in the C57BL/6J genetic background and the delayed onset of glucose intolerance in the Zucker rats, and it is proven to be a useful animal model for biomedical and pharmacological research on obesity and diabetes.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Diversity at the Locus Associated with Transcription of a Variable Surface Antigen of Pneumocystis carinii as an Index of Population Structure and Dynamics in Infected Rats

PubMed Central

Keely, Scott P.; Cushion, Melanie T.; Stringer, James R.

2003-01-01

Pneumocystis carinii expresses a surface glycoprotein called MSG. Different isoforms of MSG are encoded by a gene family spread over at least 15 telomeric sites. Only one locus, called UCS, supports the production of MSG mRNA. Previous studies showed that P. carinii populations from individual rats exhibited high degrees of diversity with respect to the MSG genes attached to the UCS locus. This diversity could have been generated primarily in the rats studied. Alternatively, the rats may have been infected by P. carinii organisms that were already different at the UCS locus. To investigate this issue, we examined the UCS locus in P. carinii from rats that had been exposed to few of the microbes at a specified time, which produced a bottleneck in the microbial population. Some of the rats with bottlenecks produced P. carinii populations in which a single MSG sequence resided at the UCS locus in 80 to 90% of the organisms, showing that P. carinii can proliferate within a rat without generating the very high levels of UCS diversity previously seen. From the degree of diversity observed in the bottlenecked populations, the maximum rate of switching appeared to be 0.01 event per generation. These data also suggest that the infectious dose is as low as one organism, that rats that share a cage readily infect each other, and that the doubling time of P. carinii in vivo is ∼3 days. In addition, we found that inoculation with 107 P. carinii organisms from a population highly heterogeneous at the UCS locus reproduced this heterogeneity. By contrast, shifts in population structure occurred in rats given 104 P. carinii organisms, suggesting that a small fraction of these proliferated. PMID:12496148

Diabetes-Induced Changes in the Alternative Splicing of the Slo Gene in Corporal Tissue

PubMed Central

Davies, Kelvin P.; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C.; Desai, Pratik; Verselis, Vytas K.; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J.

2007-01-01

Objectives Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel α-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Methods Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. Results A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Conclusions Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity. PMID:17150299

Gene transfer with a vector expressing Maxi-K from a smooth muscle-specific promoter restores erectile function in the aging rat.

PubMed

Melman, A; Biggs, G; Davies, K; Zhao, W; Tar, M T; Christ, G J

2008-03-01

Previous reports have demonstrated that gene transfer with the alpha, or pore-forming, subunit of the human Maxi-K channel (hSlo) restores the decline in erectile capacity observed in established rat models of diabetes and aging. Preliminary data from a human clinical trial also showed safety and potential efficacy in 11 men treated with the same plasmid construct expressing the Maxi-K channel. In all instances, the original plasmid was driven by the heterologous cytomegalovirus promoter which is broadly active in a wide variety of cell and tissue types. To more precisely determine the contribution of the corporal myocyte to the observed physiological effects in vivo, we report here our initial work using a distinct vector (pSMAA-hSlo) in which hSlo gene expression was driven off the mouse smooth muscle alpha-actin (SMAA) promoter. Specifically, older rats, with diminished erectile capacity, were given a single intracorporal injection with either 100 mug pVAX-hSlo or 10, 100 or 1000 mug pSMAA-hSlo, or vector or vehicle alone. Significantly increased intracavernous pressure (ICP) responses to cavernous nerve stimulation were observed for all doses of both plasmids encoding hSlo, relative to control injections. These data confirm and extend previous observations to document that smooth muscle cell-specific expression of hSlo in corporal tissue is both necessary and sufficient to restore erectile function in aging rats.

Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

PubMed Central

Puhl, Henry L.; Ikeda, Stephen R.

2008-01-01

Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

Neuroprotective effects of Bcl-2 overexpression on nerve cells of rats with acute cerebral infarction.

PubMed

Zhang, H R; Peng, J H; Zhu, G Y; Xu, R X

2015-07-13

We aimed to investigate the influence of lentiviral-mediated Bcl-2 overexpression in cerebral tissues of rats with acute cerebral infarction. Forty-five rats were randomly divided into sham, model, and treatment groups. The sham and model groups were administered a control lentiviral vector via the intracranial arteries 10 days before surgery, while the treatment group received lentivirus encoding a Bcl-2 overexpression vector. We induced cerebral artery infarction using a suture-occlusion method and analyzed the cerebral expression levels of apoptosis-related genes (caspase-3, Bax), total cerebral apoptosis, range of cerebral tissue infarction, and changes in nerve cell function after 72 h. The Bcl-2-encoding lentivirus was well expressed in rat cerebral tissues. The treatment group had significantly higher expression levels of Bcl-2 than the other two groups. After cerebral infarction, the model group had significantly increased expression levels of caspase-3 and Bax protein in cerebral tissues than the sham (P < 0.05). Expression of these apoptosis-related proteins in the treatment group was obviously lower than that in the model group (P < 0.05), but significantly higher than in the sham group (P < 0.05). Compared to sham, neuronal apoptosis levels and infarction range of cerebral tissues was increased in the model and treatment groups; however, these values in the treatment group were significantly lower than that in the model group (P < 0.05). Importantly, the treatment group had significantly decreased neurological impairment scores (P < 0.05). In conclusion, Bcl-2 over-expression can decrease neuronal apoptosis in rat cerebral tissue, and thus is neuroprotective after cerebral ischemia.

Promotion of acute-phase skin wound healing by Pseudomonas aeruginosa C4 -HSL.

PubMed

Kanno, Emi; Kawakami, Kazuyoshi; Miyairi, Shinichi; Tanno, Hiromasa; Suzuki, Aiko; Kamimatsuno, Rina; Takagi, Naoyuki; Miyasaka, Tomomitsu; Ishii, Keiko; Gotoh, Naomasa; Maruyama, Ryoko; Tachi, Masahiro

2016-12-01

A Pseudomonas aeruginosa quorum-sensing system, which produces N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-l-homoserine lactone (C 4 -HSL), regulates the virulence factors. In our previous study, 3-oxo-C 12 -HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C 4 -HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C 4 -HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague-Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti-C 4 -HSL antiserum or treated with C 4 -HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF-α synthesis. These responses were reversed, except for TNF-α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co-treated with PAO1 and anti-C 4 -HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF-α synthesis, were accelerated when C 4 -HSL was administered in the absence of PAO1. This acceleration was not affected by anti-TNF-α antibody. These results suggest that C 4 -HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Rax: Developmental and Daily Expression Patterns in the Rat Pineal Gland and Retina

PubMed Central

Rohde, Kristian; Klein, David C.; Møller, Morten; Rath, Martin F.

2011-01-01

Retina and anterior neural fold homeobox (Rax) gene encodes a transcription factor essential for vertebrate eye development. Recent microarray studies indicate that Rax is expressed in the adult rat pineal gland and retina. The present study reveals that Rax expression levels in the rat change significantly during retinal development with a peak occurring at embryonic day (E) 18, whereas Rax expression in the pineal is relatively delayed and not detectable until E20. In both tissues, Rax is expressed throughout postnatal development into adulthood. In the mature rat pineal gland, the abundance of Rax transcripts increases 2-fold during the light period with a peak occurring at dusk. These findings are consistent with the evidence that Rax is of functional importance in eye development and suggest a role of Rax in the developing pineal gland. In addition, it would appear possible that Rax contributes to phenotype maintenance in the mature retina and pineal gland and may facilitate 24-h changes in the pineal transcriptome. PMID:21749377

Characterization of rtSH3p13 gene encoding a development protein involved in vesicular traffic in spermiogenesis.

PubMed

Qiao, Yuan; Yang, Ju Xiang; Zhang, Xiao Dong L; Liu, Yu; Zhang, Jian Chao; Zong, Shu Dong; Miao, Shi Ying; Wang, Lin Fang; Koide, Samuel S

2004-06-01

A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

PubMed Central

Fishbein, Ilia; Alferiev, Ivan; Bakay, Marina; Stachelek, Stanley J.; Sobolewski, Peter; Lai, Meizan; Choi, Hoon; Chen, I.-W.; Levy, Robert J.

2012-01-01

Background Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. Methods and Results We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37°C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial AdGFP expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Conclusions Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature. PMID:18413497

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis

PubMed Central

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A.

2012-01-01

Purpose To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. Methods In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9–14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca2+-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Results Significantly lower mean activities of Ca2+-ATPase and Na+/K+ -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca2+-ATPase and Na+/K+-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. Conclusions The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication. PMID:22876134

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PubMed

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A; Geraldine, Pitchairaj

2012-01-01

To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats

PubMed Central

Zhang, Yifeng; Xu, Jiankun; Ruan, Ye Chun; Yu, Mei Kuen; O’Laughlin, Micheal; Wise, Helen; Chen, Di; Tian, Li; Shi, Dufang; Wang, Jiali; Chen, Sihui; Feng, Jian Q; Chow, Dick Ho Kiu; Xie, Xinhui; Zheng, Lizhen; Huang, Le; Huang, Shuo; Leung, Kwoksui; Lu, Na; Zhao, Lan; Li, Huafang; Zhao, Dewei; Guo, Xia; Chan, Kaiming; Witte, Frank; Chan, Hsiao Chang; Zheng, Yufeng; Qin, Ling

2017-01-01

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL-and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics. PMID:27571347

Conversion of hexadecanoic acid to hexadecenoic acid by rat Delta 6-desaturase.

PubMed

Guillou, Hervé; Rioux, Vincent; Catheline, Daniel; Thibault, Jean-Nöel; Bouriel, Monique; Jan, Sophie; D'Andrea, Sabine; Legrand, Philippe

2003-03-01

A higher content of C16:1 n-10 has recently been reported in the preputial gland of mice with a targeted disruption of the gene encoding stearoyl-CoA desaturase 1 (SCD1-/- mice) when compared with wild-type mice. This result has provided the first physiological evidence for the presence and regulation of a palmitoyl-CoA Delta 6-desaturase in mammals. To investigate the putative involvement of the known Delta 6-desaturase (FADS2) in this process, COS-7 cells expressing rat Delta 6-desaturase were incubated with C16:0. Transfected cells were able to synthesize C16:1 n-10, while nontransfected cells did not produce any C16:1 n-10. Evidence is therefore presented that the rat Delta 6-desaturase, which acts on the 18- and 24-carbon fatty acids of the n-6 and n-3 series, is also able to catalyze palmitic acid Delta 6 -desaturation.

Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro.

PubMed

Della-Maria, Julie; Gerard, Anne; Franck, Patricia; Gerard, Hubert

2002-12-30

In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells. Copyright 2002 Elsevier Science Ireland Ltd.

Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

PubMed Central

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun

2014-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. PMID:25332122

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

PubMed

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

2015-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis.

PubMed

Lysakowska, Monika Eliza; Denys, Andrzej; Sienkiewicz, Monika

2012-12-01

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren't. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ(2) test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ(2) test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.

Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell.

PubMed Central

Andersen, B; Pearse, R V; Schlegel, P N; Cichon, Z; Schonemann, M D; Bardin, C W; Rosenfeld, M G

1993-01-01

Members of the POU-domain gene family encode for transcriptional regulatory molecules that are important for terminal differentiation of several organ systems, including anterior pituitary, sensory neurons, and B lymphocytes. We have identified a POU-domain factor, referred to as sperm 1 (Sprm-1). This factor is most related to the transactivator Oct-3/4, which is expressed in the early embryo, primordial germ cells, and the egg. However, in contrast with Oct-3/4, rat Sprm-1 is selectively expressed during a 36- to 48-hr period immediately preceding meiosis I in male germ cells. Although the POU-domain of Sprm-1 is divergent from the POU-domains of Oct-1 and Oct-2, random-site-selection assay reveals that Sprm-1 preferentially binds to a specific variant of the classic octamer DNA-response element in which the optimal sequence differs from that preferred by Oct-1 and Pit-1. These data suggest that the Sprm-1 gene encodes a DNA-binding protein that may exert a regulatory function in meiotic events that are required for terminal differentiation of the male germ cell. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7902581

Assignment and expression patterns of porcine muscle-specific isoform of phosphoglycerate mutase gene.

PubMed

Qiu, Haifang; Zhao, Shuhong; Xu, Xuewen; Yerle, Martine; Liu, Bang

2008-05-01

It has been reported that the muscle-specific isoform (type M, PGAM2) of phosphoglycerate mutase (PGAM) is a housekeeping enzyme; it catalyzes the conversion of 3-phosphoglycerate into 2-phosphoglycerate in the glycolysis process to release energy. It is encoded by the Pgam2 gene. In this study, the cDNA of the porcine Pgam2 was cloned. This gene contains an open reading frame of 765 bp encoding a protein of 253 residues, and the predicted protein sequences share high similarity with other mammalians, 96% identity with humans, and 94% identity with mouse and rats. Pgam2 was mapped to SSC18q13-q21 by the RH panel. In this region, there are several QTLs, such as fat ratio, lean percentage, and diameter of muscle fiber, which affect meat production and quality. The reverse transcriptase-polymerase chain reaction revealed that the porcine Pgam2 gene was mainly expressed in the muscle tissue (skeletal muscle and cardiac muscle), and was expressed highly at skeletal muscle development stages (embryonic periods: 33, 65, and 90 days post-conception (dpc); postnatal pigs: 4 days and adult). This indicates that the Pgam2 gene plays an important role in muscle growth and development. In addition, it was demonstrated that PGAM2 locates both in cytoplasm and nuclei, and takes part in the glycometabolism process of cytoplasm and nuclei.

Epigenetic dysregulation of key developmental genes in radiation-induced rat mammary carcinomas.

PubMed

Daino, Kazuhiro; Nishimura, Mayumi; Imaoka, Tatsuhiko; Takabatake, Masaru; Morioka, Takamitsu; Nishimura, Yukiko; Shimada, Yoshiya; Kakinuma, Shizuko

2018-02-13

With the increase in the number of long-term cancer survivors worldwide, there is a growing concern about the risk of secondary cancers induced by radiotherapy. Epigenetic modifications of genes associated with carcinogenesis are attractive targets for the prevention of cancer owing to their reversible nature. To identify genes with possible changes in functionally relevant DNA methylation patterns in mammary carcinomas induced by radiation exposure, we performed microarray-based global DNA methylation and expression profiling in γ-ray-induced rat mammary carcinomas and normal mammary glands. The gene expression profiling identified dysregulation of developmentally related genes, including the downstream targets of polycomb repressive complex 2 (PRC2) and overexpression of enhancer of zeste homolog 2, a component of PRC2, in the carcinomas. By integrating expression and DNA methylation profiles, we identified ten hypermethylated and three hypomethylated genes that possibly act as tumor-suppressor genes and oncogenes dysregulated by aberrant DNA methylation; half of these genes encode developmental transcription factors. Bisulfite sequencing and quantitative PCR confirmed the dysregulation of the polycomb-regulated developmentally related transcription-factor genes Dmrt2, Hoxa7, Foxb1, Sox17, Lhx8, Gata3 and Runx1. Silencing of Hoxa7 was further verified by immunohistochemistry. These results suggest that, in radiation-induced mammary gland carcinomas, PRC2-mediated aberrant DNA methylation leads to dysregulation of developmentally related transcription-factor genes. Our findings provide clues to molecular mechanisms linking epigenetic regulation and radiation-induced breast carcinogenesis and underscore the potential of such epigenetic mechanisms as targets for cancer prevention. © 2018 UICC.

A Novel Nonparametric Approach for Neural Encoding and Decoding Models of Multimodal Receptive Fields.

PubMed

Agarwal, Rahul; Chen, Zhe; Kloosterman, Fabian; Wilson, Matthew A; Sarma, Sridevi V

2016-07-01

Pyramidal neurons recorded from the rat hippocampus and entorhinal cortex, such as place and grid cells, have diverse receptive fields, which are either unimodal or multimodal. Spiking activity from these cells encodes information about the spatial position of a freely foraging rat. At fine timescales, a neuron's spike activity also depends significantly on its own spike history. However, due to limitations of current parametric modeling approaches, it remains a challenge to estimate complex, multimodal neuronal receptive fields while incorporating spike history dependence. Furthermore, efforts to decode the rat's trajectory in one- or two-dimensional space from hippocampal ensemble spiking activity have mainly focused on spike history-independent neuronal encoding models. In this letter, we address these two important issues by extending a recently introduced nonparametric neural encoding framework that allows modeling both complex spatial receptive fields and spike history dependencies. Using this extended nonparametric approach, we develop novel algorithms for decoding a rat's trajectory based on recordings of hippocampal place cells and entorhinal grid cells. Results show that both encoding and decoding models derived from our new method performed significantly better than state-of-the-art encoding and decoding models on 6 minutes of test data. In addition, our model's performance remains invariant to the apparent modality of the neuron's receptive field.

Characteristic and intermingled neocortical circuits encode different visual object discriminations.

PubMed

Zhang, Guo-Rong; Zhao, Hua; Cook, Nathan; Svestka, Michael; Choi, Eui M; Jan, Mary; Cook, Robert G; Geller, Alfred I

2017-07-28

Synaptic plasticity and neural network theories hypothesize that the essential information for advanced cognitive tasks is encoded in specific circuits and neurons within distributed neocortical networks. However, these circuits are incompletely characterized, and we do not know if a specific discrimination is encoded in characteristic circuits among multiple animals. Here, we determined the spatial distribution of active neurons for a circuit that encodes some of the essential information for a cognitive task. We genetically activated protein kinase C pathways in several hundred spatially-grouped glutamatergic and GABAergic neurons in rat postrhinal cortex, a multimodal associative area that is part of a distributed circuit that encodes visual object discriminations. We previously established that this intervention enhances accuracy for specific discriminations. Moreover, the genetically-modified, local circuit in POR cortex encodes some of the essential information, and this local circuit is preferentially activated during performance, as shown by activity-dependent gene imaging. Here, we mapped the positions of the active neurons, which revealed that two image sets are encoded in characteristic and different circuits. While characteristic circuits are known to process sensory information, in sensory areas, this is the first demonstration that characteristic circuits encode specific discriminations, in a multimodal associative area. Further, the circuits encoding the two image sets are intermingled, and likely overlapping, enabling efficient encoding. Consistent with reconsolidation theories, intermingled and overlapping encoding could facilitate formation of associations between related discriminations, including visually similar discriminations or discriminations learned at the same time or place. Copyright © 2017 Elsevier B.V. All rights reserved.

Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides

PubMed Central

Calenda, Giulia; Tong, Yuehong; Kanika, Nirmala D.; Tar, Moses T.; Suadicani, Sylvia O.; Zhang, Xinhua; Melman, Arnold; Rougeot, Catherine

2011-01-01

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man. PMID:21784987

Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides.

PubMed

Calenda, Giulia; Tong, Yuehong; Kanika, Nirmala D; Tar, Moses T; Suadicani, Sylvia O; Zhang, Xinhua; Melman, Arnold; Rougeot, Catherine; Davies, Kelvin P

2011-10-01

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man.

Tumor necrosis factor-alpha stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons.

PubMed

Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Tumor necrosis factor-α stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

PubMed Central

Bowen, Elizabeth J.; Schmidt, Thomas W.; Firm, Christina S.; Russo, Andrew F.; Durham, Paul L.

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factorα (TNFα). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNFα stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNFα caused a coordinate increase in CGRP promoter activity. TNFα treatment activated the transcription factor NF-κB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNFα induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels. PMID:16277606

Localization and characterization of γ-glutamyl cyclotransferase in cancer cells.

PubMed

Azumi, Kaoru; Ikeda, Youhei; Takeuchi, Tomoharu; Nomura, Tsuyoshi; Sabau, Sorin V; Hamada, Jun-Ichi; Okada, Futoshi; Hosokawa, Masuo; Yokosawa, Hideyoshi

2009-01-01

Using differential display analysis, we have identified a novel rat gene whose expression is increased during tumor progression in rat mammary carcinoma cell lines. This gene is an ortholog of the human chromosome 7 open reading frame 24 gene (C7orf24) and encodes a protein of 188 amino acids with no recognized protein domains. C7orf24 has been identified as γ-glutamyl cyclotransferase (GGCT), an important enzyme functioning in glutathione homeostasis. Our Northern and Western blot analyses revealed that the GGCT gene is expressed in various normal human and tumor tissues, as well as in cancer cell lines. Among the tumor tissues tested, lung tumor tissue expressed GGCT mRNA more strongly than normal lung tissue. The GGCT protein was found to be localized in the cytoplasmic region of cultured cells, where it forms a homodimer. Analysis of various deletion mutants of the GGCT protein revealed that the region containing amino acid residues 61-120 of the protein is required for its cytoplasmic localization. The comparison of the soft agar colony formation of HBL-100 cells stably expressing GGCT with that of control HBL-100 cells revealed that GGCT does not promote colony formation, suggesting that the role it plays in lung cancer cells is not related to tumorigenesis.

Tissue-Specific 5′ Heterogeneity of PPARα Transcripts and Their Differential Regulation by Leptin

PubMed Central

Garratt, Emma S.; Vickers, Mark H.; Gluckman, Peter D.; Hanson, Mark A.

2013-01-01

The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PMID:23825665

Expression of Hormonal Carcinogenesis Genes and Related Regulatory microRNAs in Uterus and Ovaries of DDT-Treated Female Rats.

PubMed

Kalinina, T S; Kononchuk, V V; Gulyaeva, L F

2017-10-01

The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.

Genotyping and subtyping of Giardia and Cryptosporidium isolates from commensal rodents in China.

PubMed

Zhao, Z; Wang, R; Zhao, W; Qi, M; Zhao, J; Zhang, L; Li, J; Liu, A

2015-05-01

Cryptosporidium and Giardia are two important zoonotic intestinal parasites responsible for diarrhoea in humans and other animals worldwide. Rodents, as reservoirs or carriers of Cryptosporidium and Giardia, are abundant and globally widespread. In the present study, we collected 232 fecal specimens from commensal rodents captured in animal farms and farm neighbourhoods in China. We collected 33 Asian house rats, 168 brown rats and 31 house mice. 6.0% (14/232) and 8.2% (19/232) of these rodents were microscopy-positive for Giardia cysts and Cryptosporidium oocysts, respectively. All 14 Giardia isolates were identified as Giardia duodenalis assemblage G at a minimum of one or maximum of three gene loci (tpi, gdh and bg). By small subunit rRNA (SSU rRNA) gene sequencing, Cryptosporidium parvum (n = 12) and Cryptosporidium muris (n = 7) were identified. The gp60 gene encoding the 60-kDa glycoprotein was successfully amplified and sequenced in nine C. parvum isolates, all of which belonged to the IIdA15G1 subtype. Observation of the same IIdA15G1 subtype in humans (previously) and in rodents (here) suggests that rodents infected with Cryptosporidium have the potential to transmit cryptosporidiosis to humans.

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

PubMed Central

Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

1994-01-01

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

The human phospholamban gene: structure and expression.

PubMed

McTiernan, C F; Frye, C S; Lemster, B H; Kinder, E A; Ogletree-Hughes, M L; Moravec, C S; Feldman, A M

1999-03-01

Phospholamban, through modulation of sarcoplasmic reticulum calcium-ATPase activity, is a key regulator of cardiac diastolic function. Alterations in phospholamban expression may define parameters of muscle relaxation. In experimental animals, phospholamban is differentially expressed in various striated and smooth muscles, and within the four chambers of the heart. Decreased phospholamban expression within the heart during heart failure has also been observed. Furthermore, regulatory elements of mammalian phospholamban genes remain poorly defined. To extend these studies to humans, we (1) characterized phospholamban expression in various human organs, (2) isolated genomic clones encoding the human phospholamban gene, and (3) prepared human phospholamban promoter/luciferase reporter constructs and performed transient transfection assays to begin identification of regulatory elements. We observed that human ventricle and quadriceps displayed high levels of phospholamban transcripts and proteins, with markedly lower expression observed in smooth muscles, while the right atria also expressed low levels of phospholamban. The human phospholamban gene structure closely resembles that reported for chicken, rabbit, rat, and mouse. Comparison of the human to other mammalian phospholamban genes indicates a marked conservation of sequence for at least 217 bp upstream of the transcription start site, which contains conserved motifs for GATA, CP1/NFY, M-CAT-like, and E-box elements. Transient transfection assays with a series of plasmids containing deleted 5' flanking regions (between -2530 and -66 through +85) showed that sequences between -169 and the CP1-box at -93 were required for maximal promoter activity in neonatal rat cardiomyocytes. Activity of these reporters in HeLa cells was markedly lower than that observed in rat cardiomyocytes, suggesting at least a partial tissue selectivity of these reporter constructs.

Consequences of early life stress on the expression of endocannabinoid-related genes in the rat brain.

PubMed

Marco, Eva M; Echeverry-Alzate, Victor; López-Moreno, Jose Antonio; Giné, Elena; Peñasco, Sara; Viveros, Maria Paz

2014-09-01

The endocannabinoid system is involved in several physiological and pathological states including anxiety, depression, addiction and other neuropsychiatric disorders. Evidence from human and rodent studies suggests that exposure to early life stress may increase the risk of psychopathology later in life. Indeed, maternal deprivation (MD) (24 h at postnatal day 9) in rats induces behavioural alterations associated with depressive-like and psychotic-like symptoms, as well as important changes in the endocannabinoid system. As most neuropsychiatric disorders first appear at adolescence, and show remarkable sexual dimorphisms in their prevalence and severity, in the present study, we analysed the gene expression of the main components of the brain cannabinoid system in adolescent (postnatal day 46) Wistar male and female rats reared under standard conditions or exposed to MD. For this, we analysed, by real-time quantitative PCR, the expression of genes encoding for CB1 and CB2 receptors, TRPV1 and GPR55 (Cnr1, Cnr2a, Cnr2b, Trpv1, and Gpr55), for the major enzymes of synthesis, N-acyl phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL) (Nape-pld, Dagla and Daglb), and degradation, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) (Faah, Magl and Cox-2), in specific brain regions, that is, the frontal cortex, ventral and dorsal striatum, dorsal hippocampus and amygdala. In males, MD increased the genetic expression of all the genes studied within the frontal cortex, whereas in females such an increase was observed only in the hippocampus. In conclusion, the endocannabinoid system is sensitive to early life stress at the gene expression level in a sex-dependent and region-dependent manner, and these changes are already evident in the adolescent brain.

Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

PubMed

Su, S; Bird, R C

1995-09-15

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence of hG1.16 and the published rat L37 sequence. Southern-blot analysis revealed that hG1.16 exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.

Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1.

PubMed

Zelmer, Andrea; Martin, Melissa J; Gundogdu, Ozan; Birchenough, George; Lever, Rebecca; Wren, Brendan W; Luzio, J Paul; Taylor, Peter W

2010-07-01

Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C-X-C and C-C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.

The roles of gene duplication, gene conversion and positive selection in rodent Esp and Mup pheromone gene families with comparison to the Abp family.

PubMed

Karn, Robert C; Laukaitis, Christina M

2012-01-01

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining K(a)/K(s) for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with K(a)/K(s) >1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication.

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

PubMed Central

Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

2013-01-01

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

PubMed

Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

2014-01-03

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Tuberin haploinsufficiency is associated with the loss of OGG1 in rat kidney tumors

PubMed Central

Habib, Samy L; Simone, Simona; Barnes, Jeff J; Abboud, Hanna E

2008-01-01

Background Tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors. Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat. Tuberin downregulates the DNA repair enzyme 8-oxoguanine DNA-glycosylase (OGG1) with important functional consequences, compromising the ability of cells to repair damaged DNA resulting in the accumulation of the mutagenic oxidized DNA, 8-oxo-dG. Loss of function mutations of OGG1 also occurs in human kidney clear cell carcinoma and may contribute to tumorgenesis. We investigated the distribution of protein expression and the activity of OGG1 and 8-oxo-dG and correlated it with the expression of tuberin in kidneys of wild type and Eker rats and tumor from Eker rat. Results Tuberin expression, OGG1 protein expression and activity were higher in kidney cortex than in medulla or papilla in both wild type and Eker rats. On the other hand, 8-oxo-dG levels were highest in the medulla, which expressed the lowest levels of OGG1. The basal levels of 8-oxo-dG were also higher in both cortex and medulla of Eker rats compared to wild type rats. In kidney tumors from Eker rats, the loss of the second TSC2 allele is associated with loss of OGG1 expression. Immunostaining of kidney tissue shows localization of tuberin and OGG1 mainly in the cortex. Conclusion These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex. Loss of tuberin is accompanied by the loss of OGG1 contributing to tumorgenesis. In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma. PMID:18218111

Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toye, P.G.; Metzelaar, M.J.; Wijngaard, P.L.J.

1995-08-01

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryoticmore » expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5{prime} and 3{prime} regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3{prime} region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.« less

GTF2IRD2 is located in the Williams–Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix–loop–helix repeats

PubMed Central

Makeyev, Aleksandr V.; Erdenechimeg, Lkhamsuren; Mungunsukh, Ognoon; Roth, Jutta J.; Enkhmandakh, Badam; Ruddle, Frank H.; Bayarsaihan, Dashzeveg

2004-01-01

Williams–Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix–loop–helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix–loop–helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams–Beuren syndrome critical region. PMID:15243160

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats.

PubMed

Makeyev, Aleksandr V; Erdenechimeg, Lkhamsuren; Mungunsukh, Ognoon; Roth, Jutta J; Enkhmandakh, Badam; Ruddle, Frank H; Bayarsaihan, Dashzeveg

2004-07-27

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.

Effect of ginseng extract on the TGF-β1 signaling pathway in CCl4-induced liver fibrosis in rats.

PubMed

Hafez, Mohamed M; Hamed, Sherifa S; El-Khadragy, Manal F; Hassan, Zeinab K; Al Rejaie, Salim S; Sayed-Ahmed, Mohamed M; Al-Harbi, Naif O; Al-Hosaini, Khalid A; Al-Harbi, Mohamed M; Alhoshani, Ali R; Al-Shabanah, Othman A; Alsharari, Shakir Dekhal

2017-01-13

Liver diseases are major global health problems. Ginseng extract has antioxidant, immune-modulatory and anti-inflammatory activities. This study investigated the effect of ginseng extract on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Male Wistar rats were divided into four groups: control group, ginseng group, CCl 4 group and CCl 4  + ginseng group. Liver injury was induced by the intraperitoneal (I.P) injection of 3 ml/kg CCl 4 (30% in olive oil) weekly for 8 weeks. The control group was I.P injected with olive oil. The expression of genes encoding transforming growth factor beta (TGF-β), type I TGF-β receptor (TβR-1), type II TGF-β receptor (TβR-II), mothers against decapentaplegic homolog 2 (Smad2), Smad3, Smad4, matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor matrix metalloproteinase-1 (TIMP-1), Collagen 1a2 (Col1a2), Collagen 3a1 (Col3a1), interleukin-8 (IL-8) and interleukin -10 (IL-10) were measured by real-time PCR. Treatment with ginseng extract decreased hepatic fat deposition and lowered hepatic reticular fiber accumulation compared with the CCl 4 group. The CCl 4 group showed a significant increase in hepatotoxicity biomarkers and up-regulation of the expression of genes encoding TGF-β, TβR-I, TβR-II, MMP2, MMP9, Smad-2,-3, -4, and IL-8 compared with the control group. However, CCl 4 administration resulted in the significant down-regulation of IL-10 mRNA expression compared with the control group. Interestingly, ginseng extract supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl 4 . ginseng extract had an anti-fibrosis effect via the regulation of the TGF-β1/Smad signaling pathway in the CCl 4 -induced liver fibrosis model. The major target was the inhibition of the expression of TGF-β1, Smad2, and Smad3.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Rax : developmental and daily expression patterns in the rat pineal gland and retina.

PubMed

Rohde, Kristian; Klein, David C; Møller, Morten; Rath, Martin F

2011-09-01

Retina and anterior neural fold homeobox (Rax) gene encodes a transcription factor essential for vertebrate eye development. Recent microarray studies indicate that Rax is expressed in the adult rat pineal gland and retina. The present study reveals that Rax expression levels in the rat change significantly during retinal development with a peak occurring at embryonic day 18, whereas Rax expression in the pineal is relatively delayed and not detectable until embryonic day 20. In both tissues, Rax is expressed throughout postnatal development into adulthood. In the mature rat pineal gland, the abundance of Rax transcripts increases 2-fold during the light period with a peak occurring at dusk. These findings are consistent with the evidence that Rax is of functional importance in eye development and suggest a role of Rax in the developing pineal gland. In addition, it would appear possible that Rax contributes to phenotype maintenance in the mature retina and pineal gland and may facilitate 24-h changes in the pineal transcriptome. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Usher Syndrome Type III: Revised Genomic Structure of the USH3 Gene and Identification of Novel Mutations

PubMed Central

Fields, Randall R.; Zhou, Guimei; Huang, Dali; Davis, Jack R.; Möller, Claes; Jacobson, Samuel G.; Kimberling, William J.; Sumegi, Janos

2002-01-01

Usher syndrome type III is an autosomal recessive disorder characterized by progressive sensorineural hearing loss, vestibular dysfunction, and retinitis pigmentosa. The disease gene was localized to 3q25 and recently was identified by positional cloning. In the present study, we have revised the structure of the USH3 gene, including a new translation start site, 5′ untranslated region, and a transcript encoding a 232–amino acid protein. The mature form of the protein is predicted to contain three transmembrane domains and 204 residues. We have found four new disease-causing mutations, including one that appears to be relatively common in the Ashkenazi Jewish population. We have also identified mouse (chromosome 3) and rat (chromosome 2) orthologues, as well as two human paralogues on chromosomes 4 and 10. PMID:12145752

Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

NASA Astrophysics Data System (ADS)

Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

1997-09-01

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

PubMed Central

Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

1991-01-01

Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 8 PMID:1998338

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

Active form of vitamin D ameliorates non-alcoholic fatty liver disease by alleviating oxidative stress in a high-fat diet rat model.

PubMed

Zhu, Chong-Gui; Liu, Ya-Xin; Wang, Hao; Wang, Bao-Ping; Qu, Hui-Qi; Wang, Bao-Li; Zhu, Mei

2017-07-28

The purpose of this study was to determine whether treatment using the active form of vitamin D (1,25(OH) 2 D 3 ) could protect against high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats and ameliorate oxidative stress. Male Sprague-Dawley rats were divided into three groups and treated with standard chow, HFD, or HFD plus intraperitoneal injection of 1,25(OH) 2 D 3 (5 μg/kg body weight, twice per week), respectively, for 16 weeks. Serum lipid profiles, hepatic function, intrahepatic lipid, and calcium levels were determined. Hepatic histology was examined using hematoxylin/eosin, Masson's trichrome, and Oil Red O staining. Oxidative stress was assessed by measuring hepatic malondialdehyde (MDA) and F2α-isoprostane content. Expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) and downstream target genes was analyzed using quantitative RT-PCR. 1,25(OH) 2 D 3 treatment improved the serum lipid profile, reduced intrahepatic lipid levels, and attenuated hepatic steatosis and inflammation in HFD rats. Furthermore, MDA and F2α-isoprostane levels in liver tissue were reduced by 1,25(OH) 2 D 3 administration. Although 1,25(OH) 2 D 3 did not regulate the expression of Nrf2 mRNA, it did induce Nrf2 nuclear translocation. The expression of Nrf2 target genes, including Gclc, Nqo1, Sod2, and Cat, was up-regulated by 1,25(OH) 2 D 3 . We conclude that 1,25(OH) 2 D 3 protects against HFD-induced NAFLD by attenuating oxidative stress, inducing NRF2 nuclear translocation, and up-regulating the expression of genes encoding antioxidant enzymes.

Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Y.-L.; Hsueh, Y.-P., E-mail: yph@gate.sinica.edu.tw

Neurofibromin, encoded by the neurofibromatosis type 1 (NF1) gene, regulates the Ras and cAMP pathways and plays a role in proliferation and neuronal morphogenesis. The details of the molecular mechanism of neurofibromin action in these processes are still unclear. In this study, immunoprecipitation and proteomics were used to identify novel proteins from rat brain that interact with neurofibromin. Mass spectrometry analysis showed that two proteins, the collapsin response mediator protein-2 (CRMP-2) and propionyl-CoA carboxylase alpha chain (PCCA), associated with neurofibromin. Immunoprecipitation-immunoblotting analysis confirmed the interactions between neurofibromin and CRMP-2 and CRMP-4, but not CRMP-1, in rat brain. CDK5, a kinasemore » that regulates CRMP-2 in axonal outgrowth, was required for the interaction between neurofibromin and CRMP-2. Since both neurofibromin and CRMP proteins are involved in proliferation and axonal morphogenesis, these results suggest that the interaction with CRMPs contributes to the function of neurofibromin in tumorigenesis and neuronal morphogenesis.« less

Magmas Overexpression Inhibits Staurosporine Induced Apoptosis in Rat Pituitary Adenoma Cell Lines

PubMed Central

Gentilin, Erica; Minoia, Mariella; Molè, Daniela; delgi Uberti, Ettore C.; Zatelli, Maria Chiara

2013-01-01

Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development. PMID:24069394

Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

PubMed Central

2012-01-01

Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the regulatory sequences of these genes could not be confirmed. Conclusions In the thalamus, β-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies. PMID:23157480

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

PubMed

de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

2016-01-01

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

NASA Astrophysics Data System (ADS)

Zhao, Chunling; Ju, Jiyu

2015-06-01

The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

Chromosomal localization and cDNA cloning of the human DBP and TEF genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khatib, Z.A.; Inaba, T.; Valentine, M.

1994-09-15

The authors have isolated cDNA and genomic clones and determined the human chromosome positions of two genes encoding transcription factors expressed in the liver and the pituitary gland: albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). Both proteins have been identified as members of the PAR (proline and acidic amino acid-rich) subfamily of bZIP transcription factors in the rat, but human homologues have not been characterized. Using a fluorescence in situ hybridization technique, the DBP locus was assigned to chromosome 19q13, and TEF to chromosome 22q13. Each assignment was confirmed by means of human chromosome segregation in somatic cellmore » hybrids. Coding sequences of DBP and TEF, extending beyond the bZIP domain to the PAR region, were highly conserved in both human-human and interspecies comparisons. Conservation of the exon-intron boundaries of each bZIP domain-encoding exon suggested derivation from a common ancestral gene. DBP and TEF mRNAs were expressed in all tissues and cell lines examined, including brain, lung, liver, spleen, and kidney. Knowledge of the human chromosome locations of these PAR proteins will facilitate studies to assess their involvement in carcinogenesis and other fundamental biological processes. 37 refs., 5 figs., 1 tab.« less

Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

PubMed Central

Sumner, Dale R; Virdi, Amarjit S

2012-01-01

An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering. PMID:22962632

The Krüppel-like factor 4 controls biosynthesis of thyrotropin-releasing hormone during hypothalamus development.

PubMed

Pérez-Monter, Carlos; Martínez-Armenta, Miriam; Miquelajauregui, Amaya; Furlan-Magaril, Mayra; Varela-Echavarría, Alfredo; Recillas-Targa, Félix; May, Víctor; Charli, Jean-Louis; Pérez-Martínez, Leonor

2011-02-20

Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Krüppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4(-/-) mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4(-/-) mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Effect of age on the sensitivity of the rat thyroid gland to ionizing radiation

PubMed Central

Matsuu-Matsuyama, Mutsumi; Shichijo, Kazuko; Okaichi, Kumio; Kurashige, Tomomi; Kondo, Hisayoshi; Miura, Shiro; Nakashima, Masahiro

2015-01-01

Exposure to ionizing radiation during childhood is a well-known risk factor for thyroid cancer. Our study evaluated the effect of age on the radiosensitivity of rat thyroid glands. Four-week-old (4W), 7 -week-old (7W), and 8-month-old (8M) male Wistar rats were exposed to 8 Gy of whole-body X-ray irradiation. Thyroids were removed 3–72 h after irradiation, and non-irradiated thyroids served as controls. Ki67-positivity and p53 binding protein 1 (53BP1) focus formation (a DNA damage response) were evaluated via immunohistochemistry. Amounts of proteins involved in DNA damage response (p53, p53 phosphorylated at serine 15, p21), apoptosis (cleaved caspase-3), and autophagy (LC3, p62) were determined via western blotting. mRNA levels of 84 key autophagy-related genes were quantified using polymerase chain reaction arrays. Ki67-positive cells in 4W (with high proliferative activity) and 7W thyroids significantly decreased in number post-irradiation. The number of 53BP1 foci and amount of p53 phosphorylated at serine 15 increased 3 h after irradiation, regardless of age. No increase in apoptosis or in the levels of p53, p21 or cleaved caspase-3 was detected for any ages. Levels of LC3-II and p62 increased in irradiated 4W but not 8M thyroids, whereas expression of several autophagy-related genes was higher in 4W than 8M irradiated thyroids. Irradiation increased the expression of genes encoding pro-apoptotic proteins in both 4W and 8M thyroids. In summary, no apoptosis or p53 accumulation was noted, despite the expression of some pro-apoptotic genes in immature and adult thyroids. Irradiation induced autophagy in immature, but not in adult, rat thyroids. PMID:25691451

Kcna1-mutant rats dominantly display myokymia, neuromyotonia and spontaneous epileptic seizures.

PubMed

Ishida, Saeko; Sakamoto, Yu; Nishio, Takeshi; Baulac, Stéphanie; Kuwamura, Mitsuru; Ohno, Yukihiro; Takizawa, Akiko; Kaneko, Shuji; Serikawa, Tadao; Mashimo, Tomoji

2012-01-30

Mutations in the KCNA1 gene, which encodes for the α subunit of the voltage-gated potassium channel Kv1.1, cause episodic ataxia type 1 (EA1). EA1 is a dominant human neurological disorder characterized by variable phenotypes of brief episodes of ataxia, myokymia, neuromyotonia, and associated epilepsy. Animal models for EA1 include Kcna1-deficient mice, which recessively display severe seizures and die prematurely, and V408A-knock-in mice, which dominantly exhibit stress-induced loss of motor coordination. In the present study, we have identified an N-ethyl-N-nitrosourea-mutagenized rat, named autosomal dominant myokymia and seizures (ADMS), with a missense mutation (S309T) in the voltage-sensor domain, S4, of the Kcna1 gene. ADMS rats dominantly exhibited myokymia, neuromyotonia and generalized tonic-clonic seizures. They also showed cold stress-induced tremor, neuromyotonia, and motor incoordination. Expression studies of homomeric and heteromeric Kv1.1 channels in HEK cells and Xenopus oocytes, showed that, although S309T channels are transferred to the cell membrane surface, they remained non-functional in terms of their biophysical properties, suggesting a dominant-negative effect of the S309T mutation on potassium channel function. ADMS rats provide a new model, distinct from previously reported mouse models, for studying the diverse functions of Kv1.1 in vivo, as well as for understanding the pathology of EA1. Copyright © 2011 Elsevier B.V. All rights reserved.

Prenatal Nutritional Deficiency Reprogrammed Postnatal Gene Expression in Mammal Brains: Implications for Schizophrenia

PubMed Central

Xu, Jiawei; He, Guang; Zhu, Jingde; Zhou, Xinyao; St Clair, David; Wang, Teng; Xiang, Yuqian; Zhao, Qingzhu; Xing, Qinghe; Liu, Yun; Wang, Lei; Li, Qiaoli

2015-01-01

Background: Epidemiological studies have identified prenatal exposure to famine as a risk factor for schizophrenia, and animal models of prenatal malnutrition display structural and functional brain abnormalities implicated in schizophrenia. Methods: The offspring of the RLP50 rat, a recently developed animal model of prenatal famine malnutrition exposure, was used to investigate the changes of gene expression and epigenetic modifications in the brain regions. Microarray gene expression analysis was carried out in the prefrontal cortex and the hippocampus from 8 RLP50 offspring rats and 8 controls. MBD-seq was used to test the changes in DNA methylation in hippocampus depending on prenatal malnutrition exposure. Results: In the prefrontal cortex, offspring of RLP50 exhibit differences in neurotransmitters and olfactory-associated gene expression. In the hippocampus, the differentially-expressed genes are related to synaptic function and transcription regulation. DNA methylome profiling of the hippocampus also shows widespread but systematic epigenetic changes; in most cases (87%) this involves hypermethylation. Remarkably, genes encoded for the plasma membrane are significantly enriched for changes in both gene expression and DNA methylome profiling screens (p = 2.37×10–9 and 5.36×10–9, respectively). Interestingly, Mecp2 and Slc2a1, two genes associated with cognitive impairment, show significant down-regulation, and Slc2a1 is hypermethylated in the hippocampus of the RLP50 offspring. Conclusions: Collectively, our results indicate that prenatal exposure to malnutrition leads to the reprogramming of postnatal brain gene expression and that the epigenetic modifications contribute to the reprogramming. The process may impair learning and memory ability and result in higher susceptibility to schizophrenia. PMID:25522397

Remote Ischemic Preconditioning Enhances the Expression of Genes Encoding Antioxidant Enzymes and Endoplasmic Reticulum Stress-Related Proteins in Rat Skeletal Muscle.

PubMed

Park, Ui Jun; Kim, Hyoung Tae; Cho, Won Hyun; Park, Jae Hyoung; Jung, Hye Ra; Kim, Min Young

2016-12-01

Ischemic preconditioning (IPC), including remote IPC (rIPC) and direct IPC (dIPC), is a promising method to decrease ischemia-reperfusion (IR) injury. This study tested the effect of both rIPC and dIPC on the genes for antioxidant enzymes and endoplasmic reticulum (ER) stress-related proteins. Twenty rats were randomly divided into the control and study groups. In the control group (n=10), the right hind limb was sham-operated. The left hind limb (IscR) of the control group underwent IR injury without IPC. In the study group (n=10), the right hind limb received IR injury after 3 cycles of rIPC. The IscR received IR injury after 3 cycles of dIPC. Gene expression was analyzed by Quantitative real-time polymerase chain reaction from the anterior tibialis muscle. The expression of the antioxidant enzyme genes including glutathione peroxidase (GPx), superoxide dismutase (SOD) 1 and catalase (CAT) were significantly reduced in IscR compared with sham treatment. In comparison with IscR, rIPC enhanced the expression of GPx, SOD2, and CAT genes. dIPC enhanced the expression of SOD2 and CAT genes. The expression of SOD2 genes was consistently higher in rIPC than in dIPC, but the difference was only significant for SOD2. The expression of genes for ER stress-related proteins tended to be reduced in IscR in comparison with sham treatment. However, the difference was only significant for C/EBP homologous protein (CHOP). In comparison with IscR, rIPC significantly up-regulated activating transcription factor 4 and CHOP, whereas dIPC up-regulated CHOP. Both rIPC and dIPC enhanced expression of genes for antioxidant enzymes and ER stress-related proteins.

Nucleus Accumbens Core and Shell Differentially Encode Reward-Associated Cues after Reinforcer Devaluation

PubMed Central

West, Elizabeth A.

2016-01-01

Nucleus accumbens (NAc) neurons encode features of stimulus learning and action selection associated with rewards. The NAc is necessary for using information about expected outcome values to guide behavior after reinforcer devaluation. Evidence suggests that core and shell subregions may play dissociable roles in guiding motivated behavior. Here, we recorded neural activity in the NAc core and shell during training and performance of a reinforcer devaluation task. Long–Evans male rats were trained that presses on a lever under an illuminated cue light delivered a flavored sucrose reward. On subsequent test days, each rat was given free access to one of two distinctly flavored foods to consume to satiation and were then immediately tested on the lever pressing task under extinction conditions. Rats decreased pressing on the test day when the reinforcer earned during training was the sated flavor (devalued) compared with the test day when the reinforcer was not the sated flavor (nondevalued), demonstrating evidence of outcome-selective devaluation. Cue-selective encoding during training by NAc core (but not shell) neurons reliably predicted subsequent behavioral performance; that is, the greater the percentage of neurons that responded to the cue, the better the rats suppressed responding after devaluation. In contrast, NAc shell (but not core) neurons significantly decreased cue-selective encoding in the devalued condition compared with the nondevalued condition. These data reveal that NAc core and shell neurons encode information differentially about outcome-specific cues after reinforcer devaluation that are related to behavioral performance and outcome value, respectively. SIGNIFICANCE STATEMENT Many neuropsychiatric disorders are marked by impairments in behavioral flexibility. Although the nucleus accumbens (NAc) is required for behavioral flexibility, it is not known how NAc neurons encode this information. Here, we recorded NAc neurons during a training session in which rats learned that a cue predicted a specific reward and during a test session when that reward value was changed. Although encoding in the core during training predicted the ability of rats to change behavior after the reward value was altered, the NAc shell encoded information about the change in reward value during the test session. These findings suggest differential roles of the core and shell in behavioral flexibility. PMID:26818502

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

PubMed

Widjojoatmodjo, Myra N; Bogaert, Lies; Meek, Bob; Zahn, Roland; Vellinga, Jort; Custers, Jerome; Serroyen, Jan; Radošević, Katarina; Schuitemaker, Hanneke

2015-10-05

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Gene transfer of a naked plasmid (pUDK-HGF) encoding human hepatocyte growth factor attenuates skin/muscle incision and retraction-induced chronic post-surgical pain in rats.

PubMed

Hu, C; Lu, Y; Chen, X; Wu, Z; Zhang, Q

2018-05-01

Chronic post-surgical pain (CPSP) remains a major clinical problem and is often refractory to current treatments. New analgesic medications and strategies for pain relief are needed. Hepatocyte growth factor (HGF) is known to be a multi-functional growth factor and regulates various biological activities. We investigated the analgesic effect and underlying mechanism of plasmid pUDK-HGF encoding human HGF gene on CPSP induced by skin/muscle incision and retraction (SMIR) in rats. The possible changes of inflammatory factors, glial cell activation and pain sensitivity after pUDK-HGF administration were investigated by ELISA, western blot and Von Frey tests, respectively. In behavioural assays, we found that a single intramuscular or intrathecal injection of pUDK-HGF significantly attenuated mechanical hypersensitivity to von Frey stimulation of plantar ipsilateral hind paw after SMIR. Intramuscular injection of pUDK-HGF promoted blood flow and proliferation of satellite cells and inhibited inflammatory cells recruitment, collagen accumulation and expression of pronociceptive factors. Intrathecal injection of pUDK-HGF inhibited activation of spinal glial cells and production of inflammatory mediators induced by SMIR. pUDK-HGF has a strong analgesic potency and efficacy in CPSP induced by SMIR in rats. This study highlights a new strategy for the treatment of CPSP. The CPSP occurs following various surgical procedures and remains a major clinical problem due to the lack of study on the mechanisms of CPSP. Our findings provide the first evidence that pUDK-HGF attenuates SMIR-induced pain behaviuors through peripheral or central mechanisms. The peripheral analgesic effect of pUDK-HGF is associated with promoting tissue repair and inhibiting inflammatory response; furthermore, pUDK-HGF inhibits activation of spinal glial cells and overexpression of inflammatory mediators in spinal cord. Therefore, naked pUDK-HGF may be a potential therapeutic strategy for treatment of CPSP in clinic. © 2018 European Pain Federation - EFIC®.

"Triplet" polycistronic vectors encoding Gata4, Mef2c, and Tbx5 enhances postinfarct ventricular functional improvement compared with singlet vectors.

PubMed

Mathison, Megumi; Singh, Vivek P; Gersch, Robert P; Ramirez, Maricela O; Cooney, Austin; Kaminsky, Stephen M; Chiuchiolo, Maria J; Nasser, Ahmed; Yang, Jianchang; Crystal, Ronald G; Rosengart, Todd K

2014-10-01

The in situ reprogramming of cardiac fibroblasts into induced cardiomyocytes by the administration of gene transfer vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) has been shown to improve ventricular function in myocardial infarction models. The efficacy of this strategy could, however, be limited by the need for fibroblast targets to be infected 3 times--once by each of the 3 transgene vectors. We hypothesized that a polycistronic "triplet" vector encoding all 3 transgenes would enhance postinfarct ventricular function compared with use of "singlet" vectors. After validation of the polycistronic vector expression in vitro, adult male Fischer 344 rats (n=6) underwent coronary ligation with or without intramyocardial administration of an adenovirus encoding all 3 major vascular endothelial growth factor (VEGF) isoforms (AdVEGF-All6A positive), followed 3 weeks later by the administration to AdVEGF-All6A-positive treated rats of singlet lentivirus encoding G, M, or T (1×10(5) transducing units each) or the same total dose of a GMT "triplet" lentivirus vector. Western blots demonstrated that triplet and singlet vectors yielded equivalent GMT transgene expression, and fluorescence activated cell sorting demonstrated that triplet vectors were nearly twice as potent as singlet vectors in generating induced cardiomyocytes from cardiac fibroblasts. Echocardiography demonstrated that GMT triplet vectors were more effective than the 3 combined singlet vectors in enhancing ventricular function from postinfarct baselines (triplet, 37%±10%; singlet, 13%±7%; negative control, 9%±5%; P<.05). These data have confirmed that the in situ administration of G, M, and T induces postinfarct ventricular functional improvement and that GMT polycistronic vectors enhance the efficacy of this strategy. Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Characterization of rat serum amyloid A4 (SAA4): A novel member of the SAA superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rossmann, Christine; Windpassinger, Christian; Brunner, Daniela

2014-08-08

Highlights: • The full length rat SAA4 (rSAA4) mRNA was characterized by rapid amplification of cDNA ends. • rSAA4 mRNA has 1830 bases including a GA dinucleotide tandem repeat in the 5′UTR. • Three consecutive C/EBP promoter elements are crucial for transcription of rSAA4. • rSAA4 is abundantly expressed in the liver on mRNA and protein level. - Abstract: The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold duringmore » inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5′- and 3′RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/β and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.« less

Interrelationship between 3,5,3´-triiodothyronine and the circadian clock in the rodent heart.

PubMed

Peliciari-Garcia, Rodrigo Antonio; Prévide, Rafael Maso; Nunes, Maria Tereza; Young, Martin Elliot

2016-01-01

Triiodothyronine (T3) is an important modulator of cardiac metabolism and function, often through modulation of gene expression. The cardiomyocyte circadian clock is a transcriptionally based molecular mechanism capable of regulating cardiac processes, in part by modulating responsiveness of the heart to extra-cardiac stimuli/stresses in a time-of-day (TOD)-dependent manner. Although TOD-dependent oscillations in circulating levels of T3 (and its intermediates) have been established, oscillations in T3 sensitivity in the heart is unknown. To investigate the latter possibility, euthyroid male Wistar rats were treated with vehicle or T3 at distinct times of the day, after which induction of known T3 target genes were assessed in the heart (4-h later). The expression of mRNA was assessed by real-time quantitative polymerase chain reaction (qPCR). Here, we report greater T3 induction of transcript levels at the end of the dark phase. Surprisingly, use of cardiomyocyte-specific clock mutant (CCM) mice revealed that TOD-dependent oscillations in T3 sensitivity were independent of this cell autonomous mechanism. Investigation of genes encoding for proteins that affect T3 sensitivity revealed that Dio1, Dio2 and Thrb1 exhibited TOD-dependent variations in the heart, while Thra1 and Thra2 did not. Of these, Dio1 and Thrb1 were increased in the heart at the end of the dark phase. Interestingly, we observed that T3 acutely altered the expression of core clock components (e.g. Bmal1) in the rat heart. To investigate this further, rats were injected with a single dose of T3, after which expression of clock genes was interrogated at 3-h intervals over the subsequent 24-h period. These studies revealed robust effects of T3 on oscillations of both core clock components and clock-controlled genes. In summary, the current study exposed TOD-dependent sensitivity to T3 in the heart and its effects in the circadian clock genes expression.

The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection.

PubMed

Yadav, Mukesh K; Vidal, Jorge E; Go, Yoon Y; Kim, Shin H; Chae, Sung-Won; Song, Jae-Jun

2018-01-01

Objective: Streptococcus pneumoniae colonizes the nasopharynx of children, and from nasopharynx it could migrate to the middle ear and causes acute otitis media (AOM). During colonization and AOM, the pneumococcus forms biofilms. In vitro biofilm formation requires a functional LuxS/AI-2 quorum-sensing system. We investigated the role of LuxS/AI-2 signaling in pneumococcal middle ear infection, and identified the genes that are regulated by LuxS/AI-2 during pneumococcal biofilm formation. Methods: Streptococcus pneumoniae D39 wild-type and an isogenic D39Δ luxS strain were utilized to evaluate in vitro biofilm formation, and in vivo colonization and epithelial damage using a microtiter plate assay and a rat model of pneumococcal middle ear infection, respectively. Biofilm structures and colonization and epithelial damage were evaluated at the ultrastructural level by scanning electron microscopy and confocal microscopy. Microarrays were used to investigate the global genes that were regulated by LuxS/AI-2 during biofilm formation. Results: The biofilm biomass and density of D39Δ luxS were significantly ( p < 0.05) lower than those of D39 wild-type. SEM and confocal microscopy revealed that D39Δ luxS formed thin biofilms in vitro compared with D39 wild-type. The in vivo model of middle ear infection showed that D39Δ luxS resulted in ~60% less ( p < 0.05) bacterial colonization than the wild-type. SEM analysis of the rat middle ears revealed dense biofilm-like cell debris deposited on the cilia in wild-type D39-infected rats. However, little cell debris was deposited in the middle ears of the D39Δ luxS -inoculated rats, and the cilia were visible. cDNA-microarray analysis revealed 117 differentially expressed genes in D39Δ luxS compared with D39 wild-type. Among the 66 genes encoding putative proteins and previously characterized proteins, 60 were significantly downregulated, whereas 6 were upregulated. Functional annotation revealed that genes involved in DNA replication and repair, ATP synthesis, capsule biosynthesis, cell division, the cell cycle, signal transduction, transcription regulation, competence, virulence, and carbohydrate metabolism were downregulated in the absence of LuxS/AI-2. Conclusion: The S. pneumoniae LuxS/AI-2 quorum-sensing system is necessary for biofilm formation and the colonization of the ear epithelium, and caused middle ear infection in the rat model. LuxS/AI-2 regulates the expression of the genes involved in virulence and bacterial fitness during pneumococcal biofilm formation.

Antioxidative roles of sesamin, a functional lignan in sesame seed, and it's effect on lipid- and alcohol-metabolism in the liver: a DNA microarray study.

PubMed

Kiso, Yoshinobu

2004-01-01

Sesamin was orally administered to rats, and blood, bile and urine were collected periodically. Over 40% of the dose of sesamin was detected in bile as glucuronides of 2-(3, 4-methylenedioxyphenyl)-6-(3, 4-dihydroxyphenyl)-cis-dioxabicyclo[3.3.0] octane and 2-(3, 4-dihydroxyphenyl)-6-(3, 4-dihydroxyphenyl)-cis-dioxabicyclo[3.3.0] octane by 24 hr after administration. Antioxidant activities of these metabolites were compared and catechol metabolites showed strong radical scavenging activities against not only superoxide anion radical but also hydroxyl radical. It was suggested that sesamin was absorbed by the route of portal vein and metabolized to mono- or di-catechol metabolite by drug metabolizing enzymes in the liver cells. Both metabolites exhibited antioxidant activity in the liver and were finally conjugated with glucuronic acid and to excrete in bile. Sesamin can be classified as a pro-antioxidant. The profiles of gene expression of the liver in rats given sesamin or vehicle were compared. The gene expression levels of the late stage enzymes of beta-oxidation including trifunctional enzyme, acyl-CoA oxidase, bifunctional enzyme and 3-ketoacyl-CoA thiolase were significantly increased by sesamin. On the other hand, the transcription of the genes encoding the enzymes for fatty acid synthesis was decreased. Moreover, in sesamin rats, the gene expression of aldehyde dehydrogenase was increased about 3-fold, whereas alcohol dehydrogenase, liver catalase and CYP2E1 were not changed. These results suggested that sesamin ingestion regulated the transcription levels of hepatic metabolizing enzymes for lipids and alcohol.

Nitric oxide synthase gene transfer for erectile dysfunction in a rat model.

PubMed

Chancellor, M B; Tirney, S; Mattes, C E; Tzeng, E; Birder, L A; Kanai, A J; de Groat, W C; Huard, J; Yoshimura, N

2003-05-01

To determine whether over-expression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis improves erectile function, as NO is an important transmitter for genitourinary tract function, mediating smooth muscle relaxation and being essential for penile erection. The inducible form of the enzyme NOS (iNOS) was introduced into the corpus cavernosum of adult Sprague-Dawley rats (250-300 g) by injecting a solution of plasmid, adenovirus or adenovirus-transduced myoblast cells (adeno-myoblasts). Plasmid, adenovirus and adeno-myoblasts encoding the expression of the beta-galactosidase reporter gene were also injected into rats. Throughout the corpora cavernosum there was expression of beta-galactosidase after injecting each of the three solutions. Maximum staining was greatest for adeno-myoblast, then adenovirus and then plasmid. The mean (sd) basal intracavernosal pressure (ICP) of iNOS-treated animals (adenovirus and adeno-myoblast) increased to 55 (23) cmH2O, compared with naive animals with a basal ICP of 5 (6) cmH2O (P = 0.001). Stimulating the cavernosal nerve (15 Hz, 1.5 ms, 10-40 V, 1 min) resulted in a doubling of the ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals. Direct in situ measurement of NO showed the release of 1-1.3 micro mol/L in the adeno-myoblast penis. Myoblast-mediated gene therapy was more successful for delivering iNOS into the corpus cavernosum than direct adenovirus injection or plasmid transfection. Surprisingly, implanting muscle cells into the penis is not only feasible but also beneficial. Gene therapy for NOS may open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent priapism.

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

PubMed

Atta, Hussein; El-Rehany, Mahmoud; Roeb, Elke; Abdel-Ghany, Hend; Ramzy, Maggie; Gaber, Shereen

2016-02-01

Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) expression were evaluated at sacrifice one week after treatment. Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-β1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

PubMed Central

Hobbs, A A; Rosen, J M

1982-01-01

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse. Images PMID:6298707

Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

PubMed Central

Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

1992-01-01

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats.

PubMed

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-04-22

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD(+)) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD(+) precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD(+). Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD(+) availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD(+) levels.

Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

PubMed Central

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-01-01

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

Thyroid hormone activation of retinoic acid synthesis in hypothalamic tanycytes.

PubMed

Stoney, Patrick N; Helfer, Gisela; Rodrigues, Diana; Morgan, Peter J; McCaffery, Peter

2016-03-01

Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus. © 2015 Wiley Periodicals, Inc.

Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals.

PubMed

Felip, Alicia; Zanuy, Silvia; Pineda, Rafael; Pinilla, Leonor; Carrillo, Manuel; Tena-Sempere, Manuel; Gómez, Ana

2009-11-27

Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids (FNFNPFGLRF). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

PubMed Central

Loit, Evelin; Melnyk, Charles W; MacFarlane, Amanda J; Scott, Fraser W; Altosaar, Illimar

2009-01-01

Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health. PMID:19615078

TGF-β1 expression in wound healing is acutely affected by experimental malnutrition and early enteral feeding.

PubMed

Alves, Claudia Cristina; Torrinhas, Raquel Susana; Giorgi, Ricardo; Brentani, Maria Mitzi; Logullo, Angela Flavia; Waitzberg, Dan Linetzky

2014-10-01

Malnutrition is associated with the delay or failure of healing. We assessed the effect of experimental malnutrition and early enteral feeding with standard diet or diet supplemented with arginine and antioxidants on the levels of mRNA encoding growth factors in acute, open wound healing. Standardised cutaneous dorsal wounds and gastrostomies for enteral feeding were created in malnourished (M, n = 27) and eutrophic control (E, n = 30) Lewis male adult rats. Both M and E rats received isocaloric and isonitrogenous regimens with oral chow and saline (C), standard (S) or supplemented (A) enteral diets. On post-trauma day 7, mRNA levels of growth factor genes were analysed in wound granulation tissue by reverse transcription polymerase chain reaction (RT-PCR). M(C) rats had significantly lower transforming growth factor β(TGF-β1 ) mRNA levels than E(C) rats (2·58 ± 0·83 versus 3·53 ± 0·57, P < 0·01) and in comparison with M(S) and M(A) rats (4·66 ± 2·49 and 4·61 ± 2·11, respectively; P < 0·05). VEGF and KGF-7 mRNA levels were lower in M(A) rats than in E(A) rats (0·74 ± 0·16 versus 1·25 ± 0·66; and 1·07 ± 0·45 versus 1·79 ± 0·89, respectively; P≤ 0·04), but did not differ from levels in E(C) and M(C) animals. In experimental open acute wound healing, previous malnutrition decreased local mRNA levels of TGF-β1 genes, which was minimised by early enteral feeding with standard or supplemented diets. © 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Encoding changes in orbitofrontal cortex in reversal-impaired aged rats.

PubMed

Schoenbaum, Geoffrey; Setlow, Barry; Saddoris, Michael P; Gallagher, Michela

2006-03-01

Previous work in rats and primates has shown that normal aging can be associated with a decline in cognitive flexibility mediated by prefrontal circuits. For example, aged rats are impaired in rapid reversal learning, which in young rats depends critically on the orbitofrontal cortex. To assess whether aging-related reversal impairments reflect orbitofrontal dysfunction, we identified aged rats with reversal learning deficits and then recorded single units as these rats, along with unimpaired aged cohorts and young control rats, learned and reversed a series of odor discrimination problems. We found that the flexibility of neural correlates in orbitofrontal cortex was markedly diminished in aged rats characterized as reversal-impaired in initial training. In particular, although many cue-selective neurons in young and aged-unimpaired rats reversed odor preference when the odor-outcome associations were reversed, cue-selective neurons in reversal-impaired aged rats did not. In addition, outcome-expectant neurons in aged-impaired rats failed to become active during cue sampling after learning. These altered features of neural encoding could provide a basis for cognitive inflexibility associated with normal aging.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein.

PubMed

Huang, Shengbing; Song, Wei; Lin, Qishui

2005-08-01

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

PubMed Central

Grohmann, L; Brennicke, A; Schuster, W

1992-01-01

The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed. Images PMID:1454526

Downregulation of Oligodendrocyte Transcripts is Associated with Impaired Prefrontal Cortex Function in Rats

PubMed Central

Gregg, Justin R.; Herring, Nicole R.; Naydenov, Alipi V.; Hanlin, Ryan P.; Konradi, Christine

2009-01-01

Abnormalities of brain white matter and oligodendroglia are among the most consistent findings in schizophrenia (Sz) research. Various gene expression microarray studies of postmortem Sz brains showed a downregulation of myelin transcripts, while imaging and microscopy studies demonstrated decreases in prefrontal cortical (PFC) white matter volume and oligodendroglia density. Currently, the extent to which reduced oligodendrocyte markers contribute to pathophysiological domains of Sz is unknown. We exposed adolescent rats to cuprizone (CPZ), a copper chelator known to cause demyelination in mice, and examined expression of oligodendrocyte mRNA transcripts and PFC-mediated behavior. Rats on the CPZ diet showed decreased expression of mRNA transcripts encoding oligodendroglial proteins within the medial PFC, but not in the hippocampus or the striatum. These rats also displayed a specific deficit in the ability to shift between perceptual dimensions in the attentional set-shifting task, a PFC-mediated behavioral paradigm modeled after the Wisconsin Card Sorting Test (WCST). The inability to shift strategies corresponds to the deficits exhibited by Sz patients in the WCST. The results demonstrate that a reduction in oligodendrocyte markers is associated with impaired PFC-mediated behaviors. Thus, CPZ exposure of rats can serve as a model to examine the contribution of oligodendrocyte perturbation to cognitive deficits observed in Sz. PMID:19570651

cDNA cloning of the murine PEX gene implicated in X-linked hypophosphatemia and evidence for expression in bone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, L.; Desbarats, M.; Viel, J.

1996-08-15

The recently identified human PEX g ene apparently encodes for a neutral endopeptidase that is mutated in patients with X-linked hypophosphatemia. The 3{prime} and 5{prime} ends of the coding region of PEX have not been cloned, nor has the tissue expression of the gene been identified. Here we report the isolation and characterization of the complete open reading frame of the mouse Pex gene and the demonstration of its expression in bone. Mouse Pex cDNA is predicted to encode a protein of 749 amino acids with 95% identity to the available human PEX sequence and significant homology to members ofmore » the membrane-bound metalloendopeptidase family. Northern blot analysis revealed a 6.6-kb transcript in bone and in cultured osteoblasts from normal mice that was not detectable in samples from the Hyp mouse, the murine homolog of human X-linked hypophosphatemia. Pex transcripts were, however, detectable in Hyp bone by RT-PCR amplification. Of particular interest, a cDNA clone from rat incisor shows 93% sequence identity to the 5{prime} end of Pex cDNA, suggesting that Pex may be expressed in another calcified tissue, the tooth. The association of impaired mineralization of bone and teeth and disturbed renal phosphate reabsorption with altered expression of Pex suggests that the Pex gene product may play a critical role in these processes. 47 refs., 2 figs., 1 tab.« less

Interconnected network motifs control podocyte morphology and kidney function.

PubMed

Azeloglu, Evren U; Hardy, Simon V; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y; Fang, Wei; Xiong, Huabao; Neves, Susana R; Jain, Mohit R; Li, Hong; Ma'ayan, Avi; Gordon, Ronald E; He, John Cijiang; Iyengar, Ravi

2014-02-04

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

PubMed Central

Azeloglu, Evren U.; Hardy, Simon V.; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y.; Fang, Wei; Xiong, Huabao; Neves, Susana R.; Jain, Mohit R.; Li, Hong; Ma’ayan, Avi; Gordon, Ronald E.; He, John Cijiang; Iyengar, Ravi

2014-01-01

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

Substance P signalling in primary motor cortex facilitates motor learning in rats.

PubMed

Hertler, Benjamin; Hosp, Jonas Aurel; Blanco, Manuel Buitrago; Luft, Andreas Rüdiger

2017-01-01

Among the genes that are up-regulated in response to a reaching training in rats, Tachykinin 1 (Tac1)-a gene that encodes the neuropeptide Substance P (Sub P)-shows an especially strong expression. Using Real-Time RT-PCR, a detailed time-course of Tac1 expression could be defined: a significant peak occurs 7 hours after training ended at the first and second training session, whereas no up-regulation could be detected at a later time-point (sixth training session). To assess the physiological role of Sub P during movement acquisition, microinjections into the primary motor cortex (M1) contralateral to the trained paw were performed. When Sub P was injected before the first three sessions of a reaching training, effectiveness of motor learning became significantly increased. Injections at a time-point when rats already knew the task (i.e. training session ten and eleven) had no effect on reaching performance. Sub P injections did not influence the improvement of performance within a single training session, but retention of performance between sessions became strengthened at a very early stage (i.e. between baseline-training and first training session). Thus, Sub P facilitates motor learning in the very early phase of skill acquisition by supporting memory consolidation. In line with these findings, learning related expression of the precursor Tac1 occurs at early but not at later time-points during reaching training.

Decoupling kinematics and mechanics reveals coding properties of trigeminal ganglion neurons in the rat vibrissal system

PubMed Central

Bush, Nicholas E; Schroeder, Christopher L; Hobbs, Jennifer A; Yang, Anne ET; Huet, Lucie A; Solla, Sara A; Hartmann, Mitra JZ

2016-01-01

Tactile information available to the rat vibrissal system begins as external forces that cause whisker deformations, which in turn excite mechanoreceptors in the follicle. Despite the fundamental mechanical origin of tactile information, primary sensory neurons in the trigeminal ganglion (Vg) have often been described as encoding the kinematics (geometry) of object contact. Here we aimed to determine the extent to which Vg neurons encode the kinematics vs. mechanics of contact. We used models of whisker bending to quantify mechanical signals (forces and moments) at the whisker base while simultaneously monitoring whisker kinematics and recording single Vg units in both anesthetized rats and awake, body restrained rats. We employed a novel manual stimulation technique to deflect whiskers in a way that decouples kinematics from mechanics, and used Generalized Linear Models (GLMs) to show that Vg neurons more directly encode mechanical signals when the whisker is deflected in this decoupled stimulus space. DOI: http://dx.doi.org/10.7554/eLife.13969.001 PMID:27348221

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

PubMed

Gocayne, J; Robinson, D A; FitzGerald, M G; Chung, F Z; Kerlavage, A R; Lentes, K U; Lai, J; Wang, C D; Fraser, C M; Venter, J C

1987-12-01

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Effect of hypoxia on metabolic rate, core body temperature, and c-fos expression in the naked mole rat.

PubMed

Nathaniel, Thomas I; Otukonyong, Effiong; Abdellatif, Ahmed; Soyinka, Julius O

2012-10-01

Recent investigations of hypoxia physiology in the naked mole rat have opened up an interesting line of research into the basic physiological and genomic alterations that accompany hypoxia survival. The extent to which such findings connect the effect of hypoxia to metabolic rate (O₂ consumption), core body temperature (Tb), and transcripts encoding the immediate early gene product (such as c-fos) under a constant ambient temperature (Ta) is not well known. We investigated this issue in the current study. Our first sets of experiments measured Tb and metabolic rates during exposure of naked mole rats to hypoxia over a constant Ta. Hypoxia significantly decreased metabolic rates in the naked mole rat. Although core Tb also decreased during hypoxia, the effect of hypoxia in suppressing core Tb was not significant. The second series of experiments revealed that c-fos protein and mRNA expression in the hippocampus neurons (CA1) increased in naked mole rats that were repeatedly exposed to 3% O₂ for 60 min per day for 5 days when compared to normoxia. Our findings provide evidence for the up-regulation of c-fos and suppression of metabolic rate in hypoxia tolerating naked mole rats under constant ambient temperature. Metabolic suppression and c-fos upregulation constitute part of the physiological complex associated with adaptation to hypoxia. Published by Elsevier Ltd.

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

PubMed Central

2010-01-01

Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium.

PubMed Central

Langin, D; Laurell, H; Holst, L S; Belfrage, P; Holm, C

1993-01-01

The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis. Images Fig. 5 PMID:8506334

Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway

PubMed Central

Zhang, Wei; Xue, Deting; Yin, Houfa; Wang, Shengdong; Li, Chao; Chen, Erman; Hu, Dongcai; Tao, Yiqing; Yu, Jiawei; Zheng, Qiang; Gao, Xiang; Pan, Zhijun

2016-01-01

HSPA1A, which encodes cognate heat shock protein 70, plays important roles in various cellular metabolic pathways. To investigate its effects on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its expression level was compared between undifferentiated and differentiated BMSCs. Rat HSPA1A overexpression in BMSCs increased osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Moreover, it upregulated β-catenin and downregulated DKK1 and SOST. The enhanced osteogenesis due to HSPA1A overexpression was partly rescued by a Wnt/β-catenin inhibitor. Additionally, using a rat tibial fracture model, a sheet of HSPA1A-overexpressing BMSCs improved bone fracture healing, as determined by imaging and histological analysis. Taken together, these findings suggest that HSPA1A overexpression enhances osteogenic differentiation of BMSCs, partly through Wnt/β-catenin. PMID:27279016

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Last, Jerold A.; Gohil, Kishorchandra; Mathrani, Vivek C.

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkersmore » of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-{kappa}B in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.« less

Identification of two novel mutations in FAM136A and DTNA genes in autosomal-dominant familial Meniere's disease

PubMed Central

Requena, Teresa; Cabrera, Sonia; Martín-Sierra, Carmen; Price, Steven D.; Lysakowski, Anna; Lopez-Escamez, José A.

2015-01-01

Meniere's disease (MD) is a chronic disorder of the inner ear defined by sensorineural hearing loss, tinnitus and episodic vertigo, and familial MD is observed in 5–15% of sporadic cases. Although its pathophysiology is largely unknown, studies in human temporal bones have found an accumulation of endolymph in the scala media of the cochlea. By whole-exome sequencing, we have identified two novel heterozygous single-nucleotide variants in FAM136A and DTNA genes, both in a Spanish family with three affected cases in consecutive generations, highly suggestive of autosomal-dominant inheritance. The nonsense mutation in the FAM136A gene leads to a stop codon that disrupts the FAM136A protein product. Sequencing revealed two mRNA transcripts of FAM136A in lymphoblasts from patients, which were confirmed by immunoblotting. Carriers of the FAM136A mutation showed a significant decrease in the expression level of both transcripts in lymphoblastoid cell lines. The missense mutation in the DTNA gene produces a novel splice site which skips exon 21 and leads to a shorter alternative transcript. We also demonstrated that FAM136A and DTNA proteins are expressed in the neurosensorial epithelium of the crista ampullaris of the rat by immunohistochemistry. While FAM136A encodes a mitochondrial protein with unknown function, DTNA encodes a cytoskeleton-interacting membrane protein involved in the formation and stability of synapses with a crucial role in the permeability of the blood–brain barrier. Neither of these genes has been described in patients with hearing loss, FAM136A and DTNA being candidate gene for familiar MD. PMID:25305078

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes.

PubMed

Last, Jerold A; Gohil, Kishorchandra; Mathrani, Vivek C; Kenyon, Nicholas J

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkers of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-kappaB in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.

The opiorphin gene (ProL1) and its homologues function in erectile physiology.

PubMed

Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

2008-09-01

To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 microg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 microg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapic-like condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED.

The opiorphin gene (ProL1) and its homologues function in erectile physiology

PubMed Central

Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

2010-01-01

OBJECTIVE To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. MATERIALS AND METHODS We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 μg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. RESULTS Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 μg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapiclike condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. CONCLUSION All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED. PMID:18410445

PDGF-B Gene Therapy Accelerates Bone Engineering and Oral Implant Osseointegration

PubMed Central

Chang, Po-Chun; Seol, Yang-Jo; Cirelli, Joni A; Pellegrini, Gaia R.; Jin, Qiming; Franco, Lea M.; Goldstein, Steven A.; Chandler, Lois A.; Sosnowski, Barbara; Giannobile, William V.

2009-01-01

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due in part to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5×108 or 5.5×109 pfu/ml), Ad encoding luciferase (Ad-Luc; 5.5×109 pfu/ml; control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg/ml). Bone repair and osseointegration were measured via backscattered SEM, histomorphometry, microcomputed tomography, and biomechanical assessments. Further, a panel of local and systemic safety assessments was performed. Results demonstrated bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared to Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B demonstrates regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable to rhPDGF-BB protein delivery in vivo. PMID:19741730

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Nevels, Michael; Dobner, Thomas

2007-01-01

The last 50 yr of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells and athymic nude mice.

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Speiseder, Thomas; Nevels, Michael; Dobner, Thomas

2014-01-01

The last 50 years of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells, human amniotic fluid cells and athymic nude mice.

RatMap--rat genome tools and data.

PubMed

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB-Genetics at Goteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided.

RatMap—rat genome tools and data

PubMed Central

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M.; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB–Genetics at Göteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided. PMID:15608244

Gene Expression in Wilms’ Tumor Mimics the Earliest Committed Stage in the Metanephric Mesenchymal-Epithelial Transition

PubMed Central

Li, Chi-Ming; Guo, Meirong; Borczuk, Alain; Powell, Charles A.; Wei, Michelle; Thaker, Harshwardhan M.; Friedman, Richard; Klein, Ulf; Tycko, Benjamin

2002-01-01

Wilms’ tumor (WT) has been considered a prototype for arrested cellular differentiation in cancer, but previous studies have relied on selected markers. We have now performed an unbiased survey of gene expression in WTs using oligonucleotide microarrays. Statistical criteria identified 357 genes as differentially expressed between WTs and fetal kidneys. This set contained 124 matches to genes on a microarray used by Stuart and colleagues (Stuart RO, Bush KT, Nigam SK: Changes in global gene expression patterns during development and maturation of the rat kidney. Proc Natl Acad Sci USA 2001, 98:5649–5654) to establish genes with stage-specific expression in the developing rat kidney. Mapping between the two data sets showed that WTs systematically overexpressed genes corresponding to the earliest stage of metanephric development, and underexpressed genes corresponding to later stages. Automated clustering identified a smaller group of 27 genes that were highly expressed in WTs compared to fetal kidney and heterologous tumor and normal tissues. This signature set was enriched in genes encoding transcription factors. Four of these, PAX2, EYA1, HBF2, and HOXA11, are essential for cell survival and proliferation in early metanephric development, whereas others, including SIX1, MOX1, and SALL2, are predicted to act at this stage. SIX1 and SALL2 proteins were expressed in the condensing mesenchyme in normal human fetal kidneys, but were absent (SIX1) or reduced (SALL2) in cells at other developmental stages. These data imply that the blastema in WTs has progressed to the committed stage in the mesenchymal-epithelial transition, where it is partially arrested in differentiation. The WT-signature set also contained the Wnt receptor FZD7, the tumor antigen PRAME, the imprinted gene NNAT and the metastasis-associated transcription factor E1AF. PMID:12057921

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

PubMed

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

PubMed Central

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L.

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium. PMID:22479347

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

Successful nucleofection of rat adipose-derived stroma cells with Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe).

PubMed

Fülbier, Angela; Schnabel, Reinhild; Michael, Stefanie; Vogt, Peter M; Strauß, Sarah; Reimers, Kerstin; Radtke, Christine

2014-10-09

Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs. ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test. High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration. Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.

Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo.

PubMed

Mangraviti, Antonella; Tzeng, Stephany Yi; Kozielski, Kristen Lynn; Wang, Yuan; Jin, Yike; Gullotti, David; Pedone, Mariangela; Buaron, Nitsa; Liu, Ann; Wilson, David R; Hansen, Sarah K; Rodriguez, Fausto J; Gao, Guo-Dong; DiMeco, Francesco; Brem, Henry; Olivi, Alessandro; Tyler, Betty; Green, Jordan J

2015-02-24

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

PubMed

Averina, O V; Nezametdinova, V Z; Alekseeva, M G; Danilenko, V N

2012-11-01

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions.

PubMed Central

Bae, S; Seong, J; Paik, Y

2001-01-01

Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI. Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx. 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases. PMID:11171067

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Human Genomic Signatures of Brain Oscillations During Memory Encoding.

PubMed

Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

2018-05-01

Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

PubMed

Lichtensteiger, Walter; Bassetti-Gaille, Catherine; Faass, Oliver; Axelstad, Marta; Boberg, Julie; Christiansen, Sofie; Rehrauer, Hubert; Georgijevic, Jelena Kühn; Hass, Ulla; Kortenkamp, Andreas; Schlumpf, Margret

2015-04-01

The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyawaki, Izuru, E-mail: izuru-miyawaki@ds-pharma.co.jp; Tamura, Akitoshi; Matsumoto, Izumi

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague–Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance–associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNAmore » or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones. -- Highlights: ► Role of UGT and MRP2 in thyroid pathology was investigated in clobazam-treated rats. ► Clobazam induced thyroid cellular hypertrophy in SD and Gunn rats, but not EHBR rats. ► Hepatic Mrp2 gene and protein were upregulated in SD and Gunn rats, but not EHBR rats. ► Neither serum thyroid hormones (T3/T4) nor thyroid pathology changed in EHBR rats. ► Mrp2 was implied to be a key molecule in clobazam-induced thyroid pathology in rats.« less

Cocaine self-administration abolishes associative neural encoding in the nucleus accumbens necessary for higher-order learning.

PubMed

Saddoris, Michael P; Carelli, Regina M

2014-01-15

Cocaine use is often associated with diminished cognitive function, persisting even after abstinence from the drug. Likely targets for these changes are the core and shell of the nucleus accumbens (NAc), which are critical for mediating the rewarding aspects of drugs of abuse as well as supporting associative learning. To understand this deficit, we recorded neural activity in the NAc of rats with a history of cocaine self-administration or control subjects while they learned Pavlovian first- and second-order associations. Rats were trained for 2 weeks to self-administer intravenous cocaine or water. Later, rats learned a first-order Pavlovian discrimination where a conditioned stimulus (CS)+ predicted food, and a control (CS-) did not. Rats then learned a second-order association where, absent any food reinforcement, a novel cued (SOC+) predicted the CS+ and another (SOC-) predicted the CS-. Electrophysiological recordings were taken during performance of these tasks in the NAc core and shell. Both control subjects and cocaine-experienced rats learned the first-order association, but only control subjects learned the second-order association. Neural recordings indicated that core and shell neurons encoded task-relevant information that correlated with behavioral performance, whereas this type of encoding was abolished in cocaine-experienced rats. The NAc core and shell perform complementary roles in supporting normal associative learning, functions that are impaired after cocaine experience. This impoverished encoding of motivational behavior, even after abstinence from the drug, might provide a key mechanism to understand why addiction remains a chronically relapsing disorder despite repeated attempts at sobriety. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

PubMed Central

2013-01-01

Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. Conclusions A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification. PMID:24188142

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

PubMed

Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

2013-02-01

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene.

PubMed

Yao, L; Daly, W; Newland, B; Yao, S; Wang, W; Chen, B K K; Madigan, N; Windebank, A; Pandit, A

2013-12-01

Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8-T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in significantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (P<0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord.

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

PubMed Central

Jiang, Weihua; Qin, Anqi X.; Bodell, Paul W.; Baldwin, Kenneth M.; Haddad, Fadia

2012-01-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

PubMed

Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

2012-04-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

Rhythmic Diurnal Synthesis and Signaling of Retinoic Acid in the Rat Pineal Gland and Its Action to Rapidly Downregulate ERK Phosphorylation.

PubMed

Ashton, Anna; Stoney, Patrick N; Ransom, Jemma; McCaffery, Peter

2018-03-08

Vitamin A is important for the circadian timing system; deficiency disrupts daily rhythms in activity and clock gene expression, and reduces the nocturnal peak in melatonin in the pineal gland. However, it is currently unknown how these effects are mediated. Vitamin A primarily acts via the active metabolite, retinoic acid (RA), a transcriptional regulator with emerging non-genomic activities. We investigated whether RA is subject to diurnal variation in synthesis and signaling in the rat pineal gland. Its involvement in two key molecular rhythms in this gland was also examined: kinase activation and induction of Aanat, which encodes the rhythm-generating melatonin synthetic enzyme. We found diurnal changes in expression of several genes required for RA signaling, including a RA receptor and synthetic enzymes. The RA-responsive gene Cyp26a1 was found to change between day and night, suggesting diurnal changes in RA activity. This corresponded to changes in RA synthesis, suggesting rhythmic production of RA. Long-term RA treatment in vitro upregulated Aanat transcription, while short-term treatment had no effect. RA was also found to rapidly downregulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, suggesting a rapid non-genomic action which may be involved in driving the molecular rhythm in ERK1/2 activation in this gland. These results demonstrate that there are diurnal changes in RA synthesis and activity in the rat pineal gland which are partially under circadian control. These may be key to the effects of vitamin A on circadian rhythms, therefore providing insight into the molecular link between this nutrient and the circadian system.

Neuroprotective actions of perinatal choline nutrition

PubMed Central

Blusztajn, Jan Krzysztof; Mellott, Tiffany J.

2017-01-01

Choline is an essential nutrient for humans. Studies in rats and mice have shown that high choline intake during gestation or the perinatal period improves cognitive function in adulthood, prevents memory decline of old age, and protects the brain from damage and cognitive and neurological deterioration associated with epilepsy and hereditary conditions such as Down’s and Rett syndromes. These behavioral changes are accompanied by modified patterns of expression of hundreds of cortical and hippocampal genes including those encoding proteins central for learning and memory processing. The effects of choline correlate with cerebral cortical changes in DNA and histone methylation, thus suggesting an epigenomic mechanism of action of perinatal choline. PMID:23314544

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Rare Complex Mutational Profile in an ALK Inhibitor-resistant Non-small Cell Lung Cancer.

PubMed

Azzato, Elizabeth M; Deshpande, Charuhas; Aikawa, Vania; Aggarwal, Charu; Alley, Evan; Jacobs, Benjamin; Morrissette, Jennifer; Daber, Robert

2015-05-01

Testing for somatic alterations, including anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and epidermal growth factor receptor gene (EGFR) mutations, is standard practice in the diagnostic evaluation and therapeutic management of non-small cell lung cancer (NSCLC), where the results of such tests can predict response to targeted-therapy. ALK rearrangements, EGFR mutations and mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) are considered mutually exclusive in NSCLC. Herein we identified a KRAS Q22K mutation and frameshift mutations in the genes encoding serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated serine/threonine kinase (ATM) by next-generation sequencing in a patient with ALK rearrangement-positive oligo-metastatic NSCLC, whose disease progressed while on two ALK-targeted therapies. Such a complex diagnostic genetic profile has not been reported in ALK fusion-positive NSCLC. This case highlights the utility of comprehensive molecular testing in the diagnosis of NSCLC. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Effects of 90-day feeding of transgenic Bt rice TT51 on the reproductive system in male rats.

PubMed

Wang, Er Hui; Yu, Zhou; Hu, Jing; Xu, Hai Bin

2013-12-01

Rice is a staple food crop; however, the threat of pests leads to a serious decline in its output and quality. The CryAb/CryAc gene, encodes a synthetic fusion Bacillus thuringiensis (Bt) crystal protein, was introduced into rice MingHui63 to produce insect-resistant rice TT51. This study was undertaken to investigate potential unintended effects of TT51 on the reproductive system in male rats. Male rats were treated with diets containing 60% of either TT51 or MingHui63 by weight, nutritionally balanced to an AIN93G diet, for 90days. An additional negative control group of rats were fed with a rice-based AIN93G diet. Body weights, food intake, hematology, serum chemistry, serum hormone levels, sperm parameters and relative organ/body weights were measured, and gross as well as microscopic pathology were examined. No diet-related significant differences in the values of response variables were observed between rats that were fed with diet containing transgenic TT51, MingHui63 and the control in this 90-day feeding study. In addition, necropsy and histopathology examination indicated no treatment-related changes. The results from the present study indicated that TT51 does not appear to exert any effect on the reproductive system in male rats compared with MingHui63 or the control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

PubMed Central

Gocayne, J; Robinson, D A; FitzGerald, M G; Chung, F Z; Kerlavage, A R; Lentes, K U; Lai, J; Wang, C D; Fraser, C M; Venter, J C

1987-01-01

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene. Images PMID:2825184

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

PubMed Central

McPhaul, M; Berg, P

1986-01-01

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes. Images PMID:3466162

Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

PubMed

Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

2014-08-01

The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

Substance P signalling in primary motor cortex facilitates motor learning in rats

PubMed Central

Hertler, Benjamin; Hosp, Jonas Aurel; Blanco, Manuel Buitrago

2017-01-01

Among the genes that are up-regulated in response to a reaching training in rats, Tachykinin 1 (Tac1)—a gene that encodes the neuropeptide Substance P (Sub P)—shows an especially strong expression. Using Real-Time RT-PCR, a detailed time-course of Tac1 expression could be defined: a significant peak occurs 7 hours after training ended at the first and second training session, whereas no up-regulation could be detected at a later time-point (sixth training session). To assess the physiological role of Sub P during movement acquisition, microinjections into the primary motor cortex (M1) contralateral to the trained paw were performed. When Sub P was injected before the first three sessions of a reaching training, effectiveness of motor learning became significantly increased. Injections at a time-point when rats already knew the task (i.e. training session ten and eleven) had no effect on reaching performance. Sub P injections did not influence the improvement of performance within a single training session, but retention of performance between sessions became strengthened at a very early stage (i.e. between baseline-training and first training session). Thus, Sub P facilitates motor learning in the very early phase of skill acquisition by supporting memory consolidation. In line with these findings, learning related expression of the precursor Tac1 occurs at early but not at later time-points during reaching training. PMID:29281692

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

PubMed

Yin, Yan-hui; Li, Bi-chun; Wei, Guang-hui; Zhu, Cai-ye; Li, Wei; Zhang, Ya-ni; Du, Li-xin; Cao, Wen-guang

2012-05-01

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

Parental vitamin D deficiency during pregnancy is associated with increased blood pressure in offspring via Panx1 hypermethylation.

PubMed

Meems, Laura M G; Mahmud, Hasan; Buikema, Hendrik; Tost, Jörg; Michel, Sven; Takens, Janny; Verkaik-Schakel, Rikst N; Vreeswijk-Baudoin, Inge; Mateo-Leach, Irene V; van der Harst, Pim; Plösch, Torsten; de Boer, Rudolf A

2016-12-01

Vitamin D deficiency is one of the most common nutritional deficiencies worldwide. Maternal vitamin D deficiency is associated with increased susceptibility to hypertension in offspring, but the reasons for this remain unknown. The aim of this study was to determine if parental vitamin D deficiency leads to altered DNA methylation in offspring that may relate to hypertension. Male and female Sprague-Dawley rats were fed a standard or vitamin D-depleted diet. After 10 wk, nonsibling rats were mated. The conceived pups received standard chow. We observed an increased systolic and diastolic blood pressure in the offspring from depleted parents (F1-depl). Genome-wide methylation analyses in offspring identified hypermethylation of the promoter region of the Pannexin-1 (Panx1) gene in F1-depl rats. Panx1 encodes a hemichannel known to be involved in endothelial-dependent relaxation, and we demonstrated that in F1-depl rats the increase in blood pressure was associated with impaired endothelial relaxation of the large vessels, suggesting an underlying biological mechanism of increased blood pressure in children from parents with vitamin deficiency. Parental vitamin D deficiency is associated with epigenetic changes and increased blood pressure levels in offspring. Copyright © 2016 the American Physiological Society.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Estrogen receptor modulatory effects of germinated brown rice bioactives in the uterus of rats through the regulation of estrogen-induced genes

PubMed Central

Muhammad, Sani Ismaila; Maznah, Ismail; Mahmud, Rozi Bint; Saeed, Mohammed Ibrahim; Imam, Mustapha Umar; Ishaka, Aminu

2013-01-01

Purpose The expression of genes regulated by estrogen in the uterus was studied in ovariectomized (OVX) rats treated with germinated brown rice (GBR) bioactives, and compared to Remifemin or estrogen at different doses to identify the regulation of these genes in the uterus and their molecular mechanisms. Methods Rats were treated orally with GBR bioactives (phenolics), acylated steryl glucosides (ASG), γ-amino butyric acid (GABA), and γ-oryzanol (ORZ) at 100 and 200 mg/kg, Remifemin (REM) at 10 mg/kg and 20 mg/kg, or estrogen (EST) at 0.2 mg/kg. Ribonucleic acid (RNA) was extracted from the uterus, and messenger (m)RNA expression of selected genes encoding estrogen receptor-beta (ER-β), calcium-binding protein (CaBP9k), complement protein (C3), heat shock protein 70 kDa (HSP70), and interleukin (IL)-4 receptor were quantified. Similarly, serum steroid hormone concentration was monitored at 2, 4, and 8 weeks after treatments. ER-β antibody binding to the uterus sections was also studied using immunohistochemistry. Results The group treated with EST (0.2 mg/kg) upregulated ER-β, C3, and IL-4 receptor genes compared to other groups (P<0.001). GBR phenolics (200 mg/kg) treatment upregulated the ER-β gene almost to the level of the sham non-treated group. The CaBP9k gene showed upregulation in groups treated with ASG (200 mg/kg), EST (0.2 mg/kg), and ORZ (200 mg/kg) (P<0.05). Estrogen levels increased in groups treated with EST, ASG, and ORZ (200 mg/kg) compared to the OVX untreated group (P<0.05), and there was a slight non-significant decrease (P>0.05) in the progesterone levels in the OVX untreated group compared to the sham and other treated groups. There was a significant increase at 8 weeks in the level of FSH (P<0.05) in the treated groups compared to the OVX untreated group. There was no significant difference (P>0.05) in serum luteinizing hormone (LH) between the OVX untreated group and other groups. The sham and GBR phenolics treated group showed ER-β reactivity at the glandular epithelium, while the group treated with EST showed immunoreactivity at the glandular, luminal, and stromal epithelium. Conclusion GBR phenolics moderately regulate the expression of ER-β, HSP70, and IL-4 receptor genes, and gave a positive immunoreaction to ER-β antigen in the uterus. ASG regulates the expression of CaBP9k and IL-4 receptor genes, and ORZ regulates the expression of the CaBP9k gene, while GABA at 100 mg/kg regulates the expression of the HSP70 gene. GBR and its bioactives might have an effect on estrogen-regulated genes in the uterus of rats. PMID:24324328

Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: implications for gene therapy of restenosis.

PubMed

Forbes, Scott P; Alferiev, Ivan S; Chorny, Michael; Adamo, Richard F; Levy, Robert J; Fishbein, Ilia

2013-09-01

Gene therapy with viral vectors encoding for NOS enzymes has been recognized as a potential therapeutic approach for the prevention of restenosis. Optimal activity of iNOS is dependent on the intracellular availability of L-Arg and BH4 via prevention of NOS decoupling and subsequent ROS formation. Herein, we investigated the effects of separate and combined L-Arg and BH4 supplementation on the production of NO and ROS in cultured rat arterial smooth muscle and endothelial cells transduced with AdiNOS, and their impact on the antirestenotic effectiveness of AdiNOS delivery to balloon-injured rat carotid arteries. Supplementation of AdiNOS transduced endothelial and vascular smooth muscle cells with L-Arg (3.0 mM), BH4 (10 μM) and especially their combination resulted in a significant increase in NO production as measured by nitrite formation in media. Formation of ROS was dose-dependently increased following transduction with increasing MOIs of AdiNOS. Exposure of RASMC to AdiNOS tethered to meshes via a hydrolyzable cross-linker, modeling viral delivery from stents, resulted in increased ROS production, which was decreased by supplementation with BH4 but not L-Arg or L-Arg/BH4. Enhanced cell death, caused by AdiNOS transduction, was also preventable with BH4 supplementation. In the rat carotid model of balloon injury, intraluminal delivery of AdiNOS in BH4-, L-Arg-, and especially in BH4 and L-Arg supplemented animals was found to significantly enhance the antirestenotic effects of AdiNOS-mediated gene therapy. Fine-tuning of iNOS function by L-Arg and BH4 supplementation in the transduced vasculature augments the therapeutic potential of gene therapy with iNOS for the prevention of restenosis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: Implications for gene therapy of restenosis

PubMed Central

Forbes, Scott P.; Alferiev, Ivan S.; Chorny, Michael; Adamo, Richard F.; Levy, Robert J.; Fishbein, Ilia

2013-01-01

Objective Gene therapy with viral vectors encoding for NOS enzymes has been recognized as a potential therapeutic approach for the prevention of restenosis. Optimal activity of iNOS is dependent on the intracellular availability of L-Arg and BH4 via prevention of NOS decoupling and subsequent ROS formation. Herein, we investigated the effects of separate and combined L-Arg and BH4 supplementation on the production of NO and ROS in cultured rat arterial smooth muscle and endothelial cells transduced with AdiNOS, and their impact on the antirestenotic effectiveness of AdiNOS delivery to balloon-injured rat carotid arteries. Methods and Results Supplementation of AdiNOS transduced endothelial and vascular smooth muscle cells with L-Arg (3.0 mM), BH4 (10 μM) and especially their combination resulted in a significant increase in NO production as measured by nitrite formation in media. Formation of ROS was dose-dependently increased following transduction with increasing MOIs of AdiNOS. Exposure of RASMC to AdiNOS tethered to meshes via a hydrolysable cross-linker, modeling viral delivery from stents, resulted in increased ROS production, which was decreased by supplementation with BH4 but not L-Arg or L-Arg/BH4. Enhanced cell death, caused by AdiNOS transduction, was also preventable with BH4 supplementation. In the rat carotid model of balloon injury, intraluminal delivery of AdiNOS in BH4-, L-Arg-, and especially in BH4 and L-Arg supplemented animals was found to significantly enhance the antirestenotic effects of AdiNOS-mediated gene therapy. Conclusions Fine-tuning of iNOS function by L-Arg and BH4 supplementation in the transduced vasculature augments the therapeutic potential of gene therapy with iNOS for the prevention of restenosis. PMID:23958248

Saponins, especially platycodin D, from Platycodon grandiflorum modulate hepatic lipogenesis in high-fat diet-fed rats and high glucose-exposed HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; Department of Pharmaceutical Engineering, International University of Korea, Jinju; Choi, Jae Ho

AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP-1c) pathway. Saponins, particularly platycodin D, from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have a variety of pharmacological properties, including antioxidant and hepatoprotective properties. The aim of this study was to investigate the effects of CKS on hepatic lipogenesis and on the expression of genes involved in lipogenesis, and the mechanisms involved. CKS attenuated fat accumulation and the induction of the lipogenic genes encoding SREBP-1c and fatty acid synthase in the liversmore » of HFD-fed rats and in steatotic HepG2 cells. Blood biochemical analyses and histopathological examinations showed that CKS prevented liver injury. CKS and platycodin D each increased the phosphorylation of AMPK and acetyl-CoA carboxylase in HFD-fed rats and HepG2 cells. The use of specific inhibitors showed that platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells. This study demonstrates that CKS or platycodin D alone can regulate hepatic lipogenesis via an AMPK-dependent signalling pathway. - Highlights: ► CKS attenuated fat accumulation in HFD-fed rats and in steatotic HepG2 cells. ► CKS and its major component, platycodin D, inhibited the levels of SREBP-1 and FAS. ► CKS and platycodin D increased the phosphorylation of AMPK and ACC. ► Platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells.« less

Supplementing healthy rats with a high-niacin dose has no effect on muscle fiber distribution and muscle metabolic phenotype.

PubMed

Scholz, Kristen; Kynast, Anna Marie; Couturier, Aline; Mooren, Frank-Christoph; Krüger, Karsten; Most, Erika; Eder, Klaus; Ringseis, Robert

2014-08-01

It was recently shown that niacin prevents the obesity-induced type I to type II fiber switching in skeletal muscle of obese rats and favors the development of a more oxidative metabolic phenotype and thereby increases whole body utilization of fatty acids. Whether niacin also causes type II to type I fiber switching in skeletal muscle of healthy rats has not been investigated yet. Thus, the present study aimed to investigate whether niacin supplementation influences fiber distribution and metabolic phenotype of different skeletal muscles with a distinct type I-to-type II fiber ratio in healthy rats. Twenty-four male, 10-week-old Sprague-Dawley rats were randomly assigned into two groups of 12 rats each and fed either a control diet with 30 mg supplemented niacin/kg diet (control group) or a high-niacin diet with 780 mg supplemented niacin/kg diet (high-niacin group). After 27 days of treatment, the percentage number of type I fibers in rectus femoris, gastrocnemius, and tibialis anterior muscles was 5-10% greater in the niacin group than in the control group, but did not differ between groups in soleus and vastus intermedius muscles. Transcript levels of genes encoding transcription factors regulating fiber switching, fiber-specific myosin heavy chain isoforms, and proteins involved in fatty acid utilization, oxidative phosphorylation, and angiogenesis did not differ between groups. The results show that niacin has only negligible effects on fiber distribution and its regulation as well as the metabolic phenotype of skeletal muscle in healthy rats.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Rat Humanin is encoded and translated in mitochondria and is localized to the mitochondrial compartment where it regulates ROS production.

PubMed

Paharkova, Vladislava; Alvarez, Griselda; Nakamura, Hiromi; Cohen, Pinchas; Lee, Kuk-Wha

2015-09-15

Evidence for the putative mitochondrial origin of the Humanin (HN) peptide has been lacking, although its cytoprotective activity has been demonstrated in a variety of organismal and cellular systems. We sought to establish proof-of-principle for a mitochondria-derived peptide (MDP) in a rat-derived cellular system as the rat HN sequence is predicted to lack nuclear insertions of mitochondrial origin (NUMT). We found that the rat HN (Rattin; rHN) homologue is derived from the mitochondrial genome as evidenced by decreased production in Rho-0 cells, and that peptide translation occurs in the mitochondria as it is unaffected by cycloheximide. Rat HN localizes to the mitochondria in cellular subfractionation and immunohistochemical studies. Addition of a HN analogue to isolated mitochondria from rat INS-1 beta cells reduced hydrogen peroxide production by 55%. In summary, a locally bioactive peptide is derived and translated from an open reading frame (ORF) within rat mitochondrial DNA encoding 16S rRNA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genome Editing in Rats Using TALE Nucleases.

PubMed

Tesson, Laurent; Remy, Séverine; Ménoret, Séverine; Usal, Claire; Thinard, Reynald; Savignard, Chloé; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

2016-01-01

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Neurotranscriptomics: The Effects of Neonatal Stimulus Deprivation on the Rat Pineal Transcriptome

PubMed Central

Hartley, Stephen W.; Coon, Steven L.; Savastano, Luis E.; Mullikin, James C.; Fu, Cong; Klein, David C.

2015-01-01

The term neurotranscriptomics is used here to describe genome-wide analysis of neural control of transcriptomes. In this report, next-generation RNA sequencing was using to analyze the effects of neonatal (5-days-of-age) surgical stimulus deprivation on the adult rat pineal transcriptome. In intact animals, more than 3000 coding genes were found to exhibit differential expression (adjusted-p < 0.001) on a night/day basis in the pineal gland (70% of these increased at night, 376 genes changed more than 4-fold in either direction). Of these, more than two thousand genes were not previously known to be differentially expressed on a night/day basis. The night/day changes in expression were almost completely eliminated by neonatal removal (SCGX) or decentralization (DCN) of the superior cervical ganglia (SCG), which innervate the pineal gland. Other than the loss of rhythmic variation, surgical stimulus deprivation had little impact on the abundance of most genes; of particular interest, expression levels of the melatonin-synthesis-related genes Tph1, Gch1, and Asmt displayed little change (less than 35%) following DCN or SCGX. However, strong and consistent changes were observed in the expression of a small number of genes including the gene encoding Serpina1, a secreted protease inhibitor that might influence extracellular architecture. Many of the genes that exhibited night/day differential expression in intact animals also exhibited similar changes following in vitro treatment with norepinephrine, a superior cervical ganglia transmitter, or with an analog of cyclic AMP, a norepinephrine second messenger in this tissue. These findings are of significance in that they establish that the pineal-defining transcriptome is established prior to the neonatal period. Further, this work expands our knowledge of the biological process under neural control in this tissue and underlines the value of RNA sequencing in revealing how neurotransmission influences cell biology. PMID:26367423

Efficacy of vaccination with plasmid DNA encoding for HER2/neu or HER2/neu-eGFP fusion protein against prostate cancer in rats.

PubMed

Bhattachary, R; Bukkapatnam, R; Prawoko, I; Soto, J; Morgan, M; Salup, R R

2002-05-01

Despite early diagnosis and improved therapy, 31,500 men will die from prostate cancer (PC) this year. The HER2/neu oncoprotein is an important effector of cell growth found in the majority of high-grade prostatic tumors and is capable of rendering immunogenicity. The antigenicity of this oncoprotein might prove useful in the development of PC vaccines. Our goal is to prove the principle that a single DNA vaccine can provide reliable immunity against PC in the MatLyLu (MLL) translational tumor model. The parental rat MatLyLu PC cell line expresses low to moderate levels of the rat neu protein. To simulate in vivo human PC, MatLyLu cells were transfected with a truncated sequence of human HER2/neu cDNA cloned into the pCI-neo vector. This HER2/neu cDNA sequence encodes the first 433 amino acids of the extracellular domain (ECD). MatLyLu cells were also transfected with the same HER2/neu cDNA sequence cloned into the N1-terminal sequence of EGFP reporter gene to produce a fusion protein. The partial ECD sequence of HER2/neu includes five rat major histocompatibility (MHC)-II-restricted peptides with complete human-to-rat cross-species homology. The HER2/neu protein overexpression was documented by Western Blot analysis, and the expression of fusion protein was monitored by confocal microscopy and fluorimetry. Vaccination with a single injection of HER2/neu cDNA protected 50% of animals against HER2/neu-MatLyLu tumors (P < 0.01). When the tumor cells were engineered to express HER2/neu-EGFP fusion protein, the antitumor immunity was enhanced, as following vaccination with HER2/neu-EGFP cDNA, 80% of these rats rejected HER2/neu-EGFP-MatLyLu (P<0.001). Both vaccines induced HER2/neu-specific antibody titers. Rats vaccinated with EGFP-cDNA rejected 80% of EGFP-MatLyLu tumors and, interestingly, 40% of HER2/neu-MatLyLu tumors. None of the cDNA vaccines induced immunity against parental MatLyLu cells. Our data clearly demonstrate that a single injection of HER2/neu-EGFP cDNA is a very effective vaccine against PC tumors expressing the cognate tumor-associated antigen (TA). The antitumor immunity is significantly more pronounced if the tumors express xenogeneic HER2/neu-EGFP fusion protein as opposed to only the syngeneic HER2/neu oncoprotein. Our data suggests that the HER2/neu-EGFP-MatLyLu tumor is a potential animal tumor model for investigating therapeutic vaccine strategies against PC in vivo and demonstrates the limitations of a cDNA vaccine only encoding for MHC-II-restricted HER2/neu-ECD sequence peptides.

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

A new liposome-based gene delivery system targeting lung epithelial cells using endothelin antagonist.

PubMed

Allon, Nahum; Saxena, Ashima; Chambers, Carolyn; Doctor, Bhupendra P

2012-06-10

We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs. Copyright © 2011 Elsevier B.V. All rights reserved.

Selenium Pretreatment Alleviated LPS-Induced Immunological Stress Via Upregulation of Several Selenoprotein Encoding Genes in Murine RAW264.7 Cells.

PubMed

Wang, Longqiong; Jing, Jinzhong; Yan, Hui; Tang, Jiayong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Tian, Gang; Cai, Jingyi; Shang, Haiying; Zhao, Hua

2018-04-18

This study was conducted to profile selenoprotein encoding genes in mouse RAW264.7 cells upon lipopolysaccharide (LPS) challenge and integrate their roles into immunological regulation in response to selenium (Se) pretreatment. LPS was used to develop immunological stress in macrophages. Cells were pretreated with different levels of Se (0, 0.5, 1.0, 1.5, 2.0 μmol Se/L) for 2 h, followed by LPS (100 ng/mL) stimulation for another 3 h. The mRNA expression of 24 selenoprotein encoding genes and 9 inflammation-related genes were investigated. The results showed that LPS (100 ng/mL) effectively induced immunological stress in RAW264.7 cells with induced inflammation cytokines, IL-6 and TNF-α, mRNA expression, and cellular secretion. LPS increased (P < 0.05) mRNA profiles of 9 inflammation-related genes in cells, while short-time Se pretreatment modestly reversed (P < 0.05) the LPS-induced upregulation of 7 genes (COX-2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, and MCP-1) and further increased (P < 0.05) expression of IFN-β and TNF-α in stressed cells. Meanwhile, LPS decreased (P < 0.05) mRNA levels of 18 selenoprotein encoding genes and upregulated mRNA levels of TXNRD1 and TXNRD3 in cells. Se pretreatment recovered (P < 0.05) expression of 3 selenoprotein encoding genes (GPX1, SELENOH, and SELENOW) in a dose-dependent manner and increased (P < 0.05) expression of another 5 selenoprotein encoding genes (SELENOK, SELENOM, SELENOS, SELENOT, and TXNRD2) only at a high level (2.0 μmol Se/L). Taken together, LPS-induced immunological stress in RAW264.7 cells accompanied with the global downregulation of selenoprotein encoding genes and Se pretreatment alleviated immunological stress via upregulation of a subset of selenoprotein encoding genes.

Two candidate genes for two quantitative trait loci epistatically attenuate hypertension in a novel pathway.

PubMed

Chauvet, Cristina; Ménard, Annie; Deng, Alan Y

2015-09-01

Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat models of human polygenic hypertension. They influence BP physiologically via epistatic modules. Little is known about the causal genes and virtually nothing is known on modularized mechanisms governing their regulatory connections. Two genes responsible for two individual BP QTLs on rat Chromosome 18 have been identified that belong to the same epistatic module. Treacher Collins-Franceschetti syndrome 1 (Tcof1) gene is the only function candidate for C18QTL3. Haloacid dehalogenase like hydrolase domain containing 2 (Hdhd2), although a gene of previously unknown function, is C18QTL4, and encodes a newly identified phosphatase. The current work has provided the premier evidence that Hdhd2/C18QTL4 and Tcof1/C18QTL3 may be involved in polygenic hypertension. Hdhd2/C18QTL4 can regulate the function of Tcof1/C18QTL3 via de-phosphorylation, and, for the first time, furbishes a molecular mechanism in support of a genetically epistatic hierarchy between two BP QTLs, and thus authenticates the epistasis-common pathway paradigm. The pathway initiated by Hdhd2/C18QTL4 upstream of Tcof1/C18QTL3 reveals novel mechanistic insights into BP modulations. Their discovery might yield innovative therapeutic targets and diagnostic tools predicated on a novel BP cause and mechanism that is determined by a regulatory hierarchy. Optimizing the de-phosphorylation capability and its downstream target could be antihypertensive. The conceptual paradigm of an order and regulatory hierarchy may help unravel genetic and molecular relationships among certain human BP QTLs.

Substance P acting via the neurokinin-1 receptor regulates adverse myocardial remodeling in a rat model of hypertension

PubMed Central

Dehlin, Heather M.; Manteufel, Edward J.; Monroe, Andrew L.; Reimer, Michael H.; Levick, Scott P.

2013-01-01

Background Substance P is a sensory nerve neuropeptide located near coronary vessels in the heart. Therefore, substance P may be one of the first mediators released in the heart in response to hypertension, and can contribute to adverse myocardial remodeling via interactions with the neurokinin-1 receptor. We asked: 1) whether substance P promoted cardiac hypertrophy, including the expression of fetal genes known to be re-expressed during pathological hypertrophy; and 2) the extent to which substance P regulated collagen production and fibrosis. Methods and Results Spontaneously hypertensive rats (SHR) were treated with the neurokinin-1 receptor antagonist L732138 (5 mg/kg/d) from 8 to 24 weeks of age. Age-matched WKY served as controls. The gene encoding substance P, TAC1, was up-regulated as blood pressure increased in SHR. Fetal gene expression by cardiomyocytes was increased in SHR and was prevented by L732138. Cardiac fibrosis also occurred in the SHR and was prevented by L732138. Endothelin-1 was up-regulated in the SHR and this was prevented by L732138. In isolated cardiac fibroblasts, substance P transiently up-regulated several genes related to cell-cell adhesion, cell-matrix adhesion, and extracellular matrix regulation, however, no changes in fibroblast function were observed. Conclusions Substance P activation of the neurokinin-1 receptor induced expression of fetal genes related to pathological hypertrophy in the hypertensive heart. Additionally, activation of the neurokinin-1 receptor was critical to the development of cardiac fibrosis. Since no functional changes were induced in isolated cardiac fibroblasts by substance P, we conclude that substance P mediates fibrosis via up-regulation of endothelin-1. PMID:23962787

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

PubMed

Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

2007-05-01

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

mRNA and microRNA analysis reveals modulation of biochemical pathways related to addiction in the ventral tegmental area of methamphetamine self-administering rats.

PubMed

Bosch, P J; Benton, M C; Macartney-Coxson, D; Kivell, B M

2015-07-19

Methamphetamine is a highly addictive central nervous system stimulant with increasing levels of abuse worldwide. Alterations to mRNA and miRNA expression within the mesolimbic system can affect addiction-like behaviors and thus play a role in the development of drug addiction. While many studies have investigated the effects of high-dose methamphetamine, and identified neurotoxic effects, few have looked at the role that persistent changes in gene regulation play following methamphetamine self-administration. Therefore, the aim of this study was to identify RNA changes in the ventral tegmental area following methamphetamine self-administration. We performed microarray analyses on RNA extracted from the ventral tegmental area of Sprague-Dawley rats following methamphetamine self-administration training (2 h/day) and 14 days of abstinence. We identified 78 miRNA and 150 mRNA transcripts that were differentially expressed (fdr adjusted p < 0.05, absolute log2 fold change >0.5); these included genes not previously associated with addiction (miR-125a-5p, miR-145 and Foxa1), loci encoding receptors related to drug addiction behaviors and genes with previously recognized roles in addiction such as miR-124, miR-181a, DAT and Ret. This study provides insight into the effects of methamphetamine on RNA expression in a key brain region associated with addiction, highlighting the possibility that persistent changes in the expression of genes with both known and previously unknown roles in addiction occur.

The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway.

PubMed

Kanika, Nirmala Devi; Tar, Moses; Tong, Yuehong; Kuppam, Dwaraka Srinivasa Rao; Melman, Arnold; Davies, Kelvin Paul

2009-10-01

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.

The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway

PubMed Central

Kanika, Nirmala Devi; Tar, Moses; Tong, Yuehong; Kuppam, Dwaraka Srinivasa Rao; Melman, Arnold

2009-01-01

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism. PMID:19657052

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum

PubMed Central

2013-01-01

Background METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. Methods We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Results Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Conclusion Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites. PMID:23937714

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum.

PubMed

Cadet, Jean Lud; Jayanthi, Subramaniam; McCoy, Michael T; Ladenheim, Bruce; Saint-Preux, Fabienne; Lehrmann, Elin; De, Supriyo; Becker, Kevin G; Brannock, Christie

2013-08-12

METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites.

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

PubMed

Nydam, Marie L; Netuschil, Nikolai; Sanders, Erin; Langenbacher, Adam; Lewis, Daniel D; Taketa, Daryl A; Marimuthu, Arumugapradeep; Gracey, Andrew Y; De Tomaso, Anthony W

2013-01-01

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Dissociating movement from movement timing in the rat primary motor cortex.

PubMed

Knudsen, Eric B; Powers, Marissa E; Moxon, Karen A

2014-11-19

Neural encoding of the passage of time to produce temporally precise movements remains an open question. Neurons in several brain regions across different experimental contexts encode estimates of temporal intervals by scaling their activity in proportion to the interval duration. In motor cortex the degree to which this scaled activity relies upon afferent feedback and is guided by motor output remains unclear. Using a neural reward paradigm to dissociate neural activity from motor output before and after complete spinal transection, we show that temporally scaled activity occurs in the rat hindlimb motor cortex in the absence of motor output and after transection. Context-dependent changes in the encoding are plastic, reversible, and re-established following injury. Therefore, in the absence of motor output and despite a loss of afferent feedback, thought necessary for timed movements, the rat motor cortex displays scaled activity during a broad range of temporally demanding tasks similar to that identified in other brain regions. Copyright © 2014 the authors 0270-6474/14/3415576-11$15.00/0.

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

PubMed

Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

2016-12-01

Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Populations of striatal medium spiny neurons encode vibrotactile frequency in rats: modulation by slow wave oscillations

PubMed Central

Hawking, Thomas G.

2013-01-01

Dorsolateral striatum (DLS) is implicated in tactile perception and receives strong projections from somatosensory cortex. However, the sensory representations encoded by striatal projection neurons are not well understood. Here we characterized the contribution of DLS to the encoding of vibrotactile information in rats by assessing striatal responses to precise frequency stimuli delivered to a single vibrissa. We applied stimuli in a frequency range (45–90 Hz) that evokes discriminable percepts and carries most of the power of vibrissa vibration elicited by a range of complex fine textures. Both medium spiny neurons and evoked potentials showed tactile responses that were modulated by slow wave oscillations. Furthermore, medium spiny neuron population responses represented stimulus frequency on par with previously reported behavioral benchmarks. Our results suggest that striatum encodes frequency information of vibrotactile stimuli which is dynamically modulated by ongoing brain state. PMID:23114217

Joint capsule treatment with enkephalin-encoding HSV-1 recombinant vector reduces inflammatory damage and behavioural sequelae in rat CFA monoarthritis.

PubMed

Lu, Ying; McNearney, Terry A; Wilson, Steven P; Yeomans, David C; Westlund, Karin N

2008-03-01

This study assessed enkephalin expression induced by intra-articular application of recombinant, enkephalin-encoding herpes virus (HSV-1) and the impact of expression on nociceptive behaviours and synovial lining inflammation in arthritic rats. Replication-conditional HSV-1 recombinant vectors with cDNA encoding preproenkephalin (HSV-ENK), or control transgene beta-galactosidase cDNA (HSV-beta-gal; control) were injected into knee joints with complete Freund's adjuvant (CFA). Joint temperatures, circumferences and nociceptive behaviours were monitored on days 0, 7, 14 and 21 post CFA and vector treatments. Lumbar (L4-6) dorsal root ganglia (DRG) and spinal cords were immunostained for met-enkephalin (met-ENK), beta-gal, HSV-1 proteins and Fos. Joint tissues were immunostained for met-ENK, HSV-1 proteins, and inflammatory mediators Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) and cyclo-oxygenase-2, or stained with haematoxylin and eosin for histopathology. Compared to exuberant synovial hypertrophy and inflammatory cell infiltration seen in arthritic rats treated with CFA only or CFA and HSV-beta-gal, the CFA- and HSV-ENK-treated arthritic rats had: (i) striking preservation of synovial membrane cytoarchitecture with minimal inflammatory cell infiltrates; (ii) significantly improved nociceptive behavioural responses to mechanical and thermal stimuli; (iii) normalized Fos staining in lumbar dorsal horn; and (iv) significantly increased met-ENK staining in ipsilateral synovial tissue, lumbar DRG and spinal cord. The HSV-1 and transgene product expression were confined to ipsilateral lumbar DRG (HSV-1, met-ENK, beta-gal). Only transgene product (met-ENK and beta-gal) was seen in lumbar spinal cord sections. Targeted delivery of enkephalin-encoding HSV-1 vector generated safe, sustained opioid-induced analgesia with protective anti-inflammatory blunting in rat inflammatory arthritis.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

A G-protein-activated inwardly rectifying K+ channel (GIRK4) from human hippocampus associates with other GIRK channels.

PubMed

Spauschus, A; Lentes, K U; Wischmeyer, E; Dissmann, E; Karschin, C; Karschin, A

1996-02-01

Transcripts of a gene, GIRK4, that encodes for a 419-amino-acid protein and shows high structural similarity to other subfamily members of G-protein-activated inwardly rectifying K+ channels (GIRK) have been identified in the human hippocampus. When expressed in Xenopus oocytes, GIRK4 yielded functional GIRK channels with activity that was enhanced by the stimulation of coexpressed serotonin 1A receptors. GIRK4 potentiated basal and agonist-induced currents mediated by other GIRK channels, possibly because of channel heteromerization. Despite the structural similarity to a putative rat KATP channel, no ATP sensitivity or KATP-typical pharmacology was observed for GIRK4 alone or GIRK4 transfected in conjunction with other GIRK channels in COS-7 cells. In rat brain, GIRK4 is expressed together with three other subfamily members, GIRK1-3, most likely in identical hippocampal neurons. Thus, heteromerization or an unknown molecular interaction may cause the physiological diversity observed within this class of K+ channels.

Interrelationship between 3,5,3′-triiodothyronine and the circadian clock in the rodent heart

PubMed Central

Peliciari-Garcia, Rodrigo Antonio; Prévide, Rafael Maso; Nunes, Maria Tereza; Young, Martin Elliot

2017-01-01

Triiodothyronine (T3) is an important modulator of cardiac metabolism and function, often through modulation of gene expression. The cardiomyocyte circadian clock is a transcriptionally-based molecular mechanism capable of regulating cardiac processes, in part by modulating responsiveness of the heart to extra-cardiac stimuli/stresses in a time-of-day- (TOD) dependent manner. Although TOD-dependent oscillations in circulating levels of T3 (and its intermediates) have been established, whether oscillations in T3 sensitivity in the heart occur is unknown. To investigate the latter possibility, euthyroid male Wistar rats were treated with vehicle or T3 at distinct times of the day, after which induction of known T3 target genes were assessed in the heart (4-h later). The expression of mRNA was assessed by Real-Time qPCR. Here, we report greater T3 induction of transcript levels at the end of the dark phase. Surprisingly, use of cardiomyocyte-specific clock mutant (CCM) mice revealed that TOD-dependent oscillations in T3 sensitivity were independent of this cell autonomous mechanism. Investigation of genes encoding for proteins that affect T3 sensitivity revealed that Dio1, Dio2, and Thrb1 exhibited TOD-dependent variations in the heart, while Thra1 and Thra2 did not. Of these, Dio1 and Thrb1 were increased in the heart at the end of the dark phase. Interestingly, we observed that T3 acutely altered the expression of core clock components (e.g., Bmal1) in the rat heart. To investigate this further, rats were injected with a single dose of T3, after which expression of clock genes were interrogated at 3-h intervals over the subsequent 24h-period. These studies revealed robust effects of T3 on oscillations of both core clock components and clock-controlled genes. In summary, the current study exposed time-of-day-dependent rhythms in cardiac T3 sensitivity, and that T3 alters the circadian clock in the heart. PMID:27661292

Tachykinin receptor and neutral endopeptidase gene expression in the rat uterus: characterization and regulation in response to ovarian steroid treatment.

PubMed

Pinto, F M; Armesto, C P; Magraner, J; Trujillo, M; Martín, J D; Candenas, M L

1999-06-01

Tachykinin neuropeptides, such as substance P, are localized to a population of sensory fibers that innervate the mammalian female reproductive tract. In the present study, we have characterized tachykinin NK1 receptor (NK1R), NK2 receptor (NK2R), and NK3 receptor (NK3R) gene expression by semiquantitative RT-PCR in uteri from ovariectomized rats and studied their regulation in response to 17beta-estradiol (E2), progesterone (P4), or a combination of both. In addition, we analyzed the expression and regulation of the neutral endopeptidase 24.11 (NEP), the most important enzyme involved in tachykinin degradation in the rat uterus. In uteri from control (olive oil-treated) rats, RT-PCR assays revealed single bands corresponding to the expected product sizes encoding complementary DNA for NK1R (232 bp), NK2R (491 bp), NK3R (325 bp), and NEP (221 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. Compared with control rats, NK1R messenger RNA (mRNA) was increased by 2-fold in uteri from rats treated with E2, was decreased by 3.3-fold in rats treated with P4, and was decreased by 1.8-fold in rats treated with both E2 and P4. Uterine NK2R mRNA levels were not altered by any steroid treatment. E2 treatment decreased by 15-fold NK3R mRNA. P4 was without effect if administered alone and did not influence the E2-induced decrease in NK3R mRNA. NEP mRNA levels were about 4-fold lower in E2-treated than in P4-treated rats. Functional studies were carried out in uteri from E2- or P4-treated ovariectomized rats to characterize the contractile response evoked by the selective tachykinin receptor agonists [Sar9Met(O2)11]substance P (NK1R selective), [Nle10]NKA-(4-10) (NK2R selective), and [MePhe7]NKB (NK3R selective) in the presence of the NEP inhibitor phosphoramidon (1 microM). A marked correlation was observed between the magnitude of the contractile response to each agonist and the level of expression determined by RT-PCR for each tachykinin receptor. The present findings show that tachykinin NK1R, NK2R, NK3R, and NEP are expressed in the rat uterus and that ovarian steroids differentially regulate their expression.

Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat[W][OA

PubMed Central

Mugford, Sam T.; Louveau, Thomas; Melton, Rachel; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Tsurushima, Tetsu; Honkanen, Suvi; Rosser, Susan J.; Lomonossoff, George P.; Osbourn, Anne

2013-01-01

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis. PMID:23532069

Myelin-oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pham-Dinh, D.; Dautigny, A.; Mattei, M.G.

1993-09-01

Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. The authors have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) andmore » B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. The authors colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC. 38 refs., 6 figs.« less

Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

PubMed

Paganelli, Fernanda L; Huebner, Johannes; Singh, Kavindra V; Zhang, Xinglin; van Schaik, Willem; Wobser, Dominique; Braat, Johanna C; Murray, Barbara E; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L

2016-07-15

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Na+-glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow.

PubMed

Sabino-Silva, R; Freitas, H S; Lamers, M L; Okamoto, M M; Santos, M F; Machado, U F

2009-03-01

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

[Vasopressin (ADH)].

PubMed

Hirai, A; Uchida, D; Yoshida, S

1992-12-01

Vasopressin is thought to play an important role, not only in the metabolism of water and electrolytes, but also in the regulation of renal hemodynamics. This year, great progress has been achieved in molecular biology of vasopressin receptors. First, the cloning of a complementary DNA, encoding the rat liver V1a arginine vasopressin receptor, was reported. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA, corresponding to the cDNA, is distributed in rat tissues, known to contain V1a receptors. Second, the cloning of a complementary DNA encoding the rat kidney V2 arginine vasopressin receptor was also successful. The kidney cDNA encodes a protein with a transmembrane topography characteristic of G protein-coupled receptors. The receptor messenger RNA is detected only in the kidney. Last year, an orally active and specific vasopressin V1 receptor antagonist, OPC-21268 was first reported. The i.v. or p.o. administration of OPC-21268 dose-dependently inhibited vasopressin-induced vasoconstriction, while that induced by angiotensin II was not affected. OPC-21268 may have clinical potentials in certain hypertensive cardiovascular disorders. In addition, an orally active and specific vasopressin V2 receptor antagonist, OPC-31260 was also reported. Oral administration of OPC-31260 inhibited antidiuretic action of arginine vasopressin. OPC-31260 is thought to be useful in the treatment of certain disorders, such as the syndrome of inappropriate secretion of ADH (SIADH).

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

PubMed Central

Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

1989-01-01

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Genome-Wide Architecture of Disease Resistance Genes in Lettuce

PubMed Central

Christopoulou, Marilena; Wo, Sebastian Reyes-Chin; Kozik, Alex; McHale, Leah K.; Truco, Maria-Jose; Wroblewski, Tadeusz; Michelmore, Richard W.

2015-01-01

Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes. PMID:26449254

Sustained viral gene delivery from a micro-fibrous, elastomeric cardiac patch to the ischemic rat heart.

PubMed

Gu, Xinzhu; Matsumura, Yasumoto; Tang, Ying; Roy, Souvik; Hoff, Richard; Wang, Bing; Wagner, William R

2017-07-01

Biodegradable and elastomeric patches have been applied to the surface of infarcted hearts as temporary mechanical supports to effectively alter adverse left ventricular remodeling processes. In this report, recombinant adeno-associated virus (AAV), known for its persistent transgene expression and low pathogenicity, was incorporated into elastomeric polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU) and processed by electrospinning into two formats (solid fibers and core-sheath fibers) designed to influence the controlled release behavior. The extended release of AAV encoding green fluorescent protein (GFP) was assessed in vitro. Sustained and localized viral particle delivery was achieved over 2 months in vitro. The biodegradable cardiac patches with or without AAV-GFP were implanted over rat left ventricular lesions three days following myocardial infarction to evaluate the transduction effect of released viral vectors. AAV particles were directly injected into the infarcted hearts as a control. Cardiac function and remodeling were significantly improved for 12 weeks after patch implantation compared to AAV injection. More GFP genes was expressed in the AAV patch group than AAV injection group, with both α-SMA positive cells and cardiac troponin T positive cells transduced in the patch group. Overall, the extended release behavior, prolonged transgene expression, and elastomeric mechanical properties make the AAV-loaded scaffold an attractive option for cardiac tissue engineering where both gene delivery and appropriate mechanical support are desired. Copyright © 2017. Published by Elsevier Ltd.

The use of SHP-2 gene transduced bone marrow mesenchymal stem cells to promote osteogenic differentiation and bone defect repair in rat.

PubMed

Fan, Dapeng; Liu, Shen; Jiang, Shichao; Li, Zhiwei; Mo, Xiumei; Ruan, Hongjiang; Zou, Gang-Ming; Fan, Cunyi

2016-08-01

Bone tissue engineering is a promising approach for bone regeneration, in which growth factors play an important role. The tyrosine phosphatase Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is essential for the differentiation, proliferation and metabolism of osteoblasts. However, SHP-2 has never been systematically studied for its effect in osteogenesis. We predicted that overexpression of SHP-2 could promote bone marrow-derived mesenchymal stem cell (BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation, determined using the following study groups: (1) BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid (BMSCs/SHP-2/OL); (2) BMSCs transduced with SHP-2 (BMSCs/-SHP-2); (3) BMSCs induced with osteoblast-inducing liquid (BMSCs/OL) and (4) pure BMSCs. Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis, western blot analysis, alkaline phosphatase activity and alizarin red S staining. For in vivo assessment, cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks. Following euthanasia, skull samples were explanted for osteogenic evaluation, including micro-computed tomography measurement, histology and immunohistochemistry staining. SHP-2 and upregulation of its gene promoted BMSC osteogenic differentiation and therefore represents a potential new therapeutic approach to bone repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1871-1881, 2016. © 2016 Wiley Periodicals, Inc.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Incentive memory: evidence the basolateral amygdala encodes and the insular cortex retrieves outcome values to guide choice between goal-directed actions.

PubMed

Parkes, Shauna L; Balleine, Bernard W

2013-05-15

Choice between goal-directed actions is determined by the relative value of their consequences. Such values are encoded during incentive learning and later retrieved to guide performance. Although the basolateral amygdala (BLA) and the gustatory region of insular cortex (IC) have been implicated in these processes, their relative contribution is still a matter of debate. Here we assessed whether these structures interact during incentive learning and retrieval to guide choice. In these experiments, rats were trained on two actions for distinct outcomes after which one of the two outcomes was devalued by specific satiety immediately before a choice extinction test. We first confirmed that, relative to appropriate controls, outcome devaluation recruited both the BLA and IC based on activation of the immediate early gene Arc; however, we found that infusion of the NMDAr antagonist ifenprodil into the BLA only abolished outcome devaluation when given before devaluation. In contrast, ifenprodil infusion into the IC was effective whether made before devaluation or test. We hypothesized that the BLA encodes and the IC retrieves incentive value for choice and, to test this, developed a novel sequential disconnection procedure. Blocking NMDAr activation unilaterally in the BLA before devaluation and then contralaterally in the IC before test abolished selective devaluation. In contrast, reversing the order of these infusions left devaluation intact. These results confirm that the BLA and IC form a circuit mediating the encoding and retrieval of outcome values, with the BLA encoding and the IC retrieving such values to guide choice.

Glutamine reduces myocardial cell apoptosis in a rat model of sepsis by promoting expression of heat shock protein 90.

PubMed

Li, Wanxia; Tao, Shaoyu; Wu, Qinghua; Wu, Tao; Tao, Ran; Fan, Jun

2017-12-01

Myocardial cell injury and cardiac myocyte apoptosis are associated with sepsis. Glutamine (Gln) has been reported to repair myocardial cell injury. The aim of this study was to explore the role of Gln on cardiac myocytes in a cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Following induction of sepsis in a CLP rat model, viral encoding heat shock protein 90 (Hsp90) gene and Hsp90dsDNA were designed to express and knockdown Hsp90, respectively. Rat cardiac tissues were examined histologically, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, Hsp90, p53 upregulated modulator of apoptosis, and p53 was measured by western blotting and real-time polymerase chain reaction. Caspase-3, caspase-8, and caspase-9 were detected by enzyme-linked immunosorbent assay. Rat cardiac myocyte damage induced by CLP was reduced by Gln treatment and Hsp90 overexpression, and these changes were reversed by Hsp90 knockdown. Bcl-2 expression, Bcl-2-associated X protein, p53, p53 upregulated modulator of apoptosis, caspase-8, caspase-9, and caspase-3 activities were significantly upregulated in the CLP model, which were reduced by Gln treatment and Hsp90 overexpression. Gln reduced apoptosis of cardiac myocytes in a rat model of sepsis, by promoting Hsp90 expression. Further studies are needed to determine the possible therapeutic action of Gln in sepsis in human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Indole, a Signaling Molecule Produced by the Gut Microbiota, Negatively Impacts Emotional Behaviors in Rats

PubMed Central

Jaglin, Mathilde; Rhimi, Moez; Philippe, Catherine; Pons, Nicolas; Bruneau, Aurélia; Goustard, Bénédicte; Daugé, Valérie; Maguin, Emmanuelle; Naudon, Laurent; Rabot, Sylvie

2018-01-01

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability. PMID:29686603

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

A gene catalogue of the Sprague-Dawley rat gut metagenome.

PubMed

Pan, Hudan; Guo, Ruijin; Zhu, Jie; Wang, Qi; Ju, Yanmei; Xie, Ying; Zheng, Yanfang; Wang, Zhifeng; Li, Ting; Liu, Zhongqiu; Lu, Linlin; Li, Fei; Tong, Bin; Xiao, Liang; Xu, Xun; Li, Runze; Yuan, Zhongwen; Yang, Huanming; Wang, Jian; Kristiansen, Karsten; Jia, Huijue; Liu, Liang

2018-05-01

Laboratory rats such as the Sprague-Dawley (SD) rats are an important model for biomedical studies in relation to human physiological or pathogenic processes. Here we report the first catalog of microbial genes in fecal samples from Sprague-Dawley rats. The catalog was established using 98 fecal samples from 49 SD rats, divided in 7 experimental groups, and collected at different time points 30 days apart. The established gene catalog comprises 5,130,167 non-redundant genes with an average length of 750 bp, among which 64.6% and 26.7% were annotated to phylum and genus levels, respectively. Functionally, 53.1%, 21.8%,and 31% of the genes could be annotated to KEGG orthologous groups, modules, and pathways, respectively. A comparison of rat gut metagenome catalogue with human or mouse revealed a higher pairwise overlap between rats and humans (2.47%) than between mice and humans (1.19%) at the gene level. Ninety-seven percent of the functional pathways in the human catalog were present in the rat catalogue, underscoring the potential use of rats for biomedical research.

Korean Red Ginseng Up-regulates C21-Steroid Hormone Metabolism via Cyp11a1 Gene in Senescent Rat Testes

PubMed Central

Kim, In-Hye; Kim, Si-Kwan; Kim, Eun-Hye; Kim, Sung-Won; Sohn, Sang-Hyun; Lee, Soo Cheol; Choi, Sangdun; Pyo, Suhkneung; Rhee, Dong-Kwon

2011-01-01

Ginseng (Panax ginseng Meyer) has been shown to have anti-aging effects in animal and clinical studies. However, the molecular mechanisms by which ginseng exerts these effects remain unknown. Here, the anti-aging effect of Korean red ginseng (KRG) in rat testes was examined by system biology analysis. KRG water extract prepared in feed pellets was administered orally into 12 month old rats for 4 months, and gene expression in testes was determined by microarray analysis. Microarray analysis identified 33 genes that significantly changed. Compared to the 2 month old young rats, 13 genes (Rps9, Cyp11a1, RT1-A2, LOC365778, Sv2b, RGD1565959, RGD1304748, etc.) were up-regulated and 20 genes (RT1-Db1, Cldn5, Svs5, Degs1, Vdac3, Hbb, LOC684355, Svs5, Tmem97, Orai1, Insl3, LOC497959, etc.) were down-regulated by KRG in the older rats. Ingenuity Pathway Analysis of untreated aged rats versus aged rats treated with KRG showed that the affected most was Cyp11a1, responsible for C21-steroid hormone metabolism, and the top molecular and cellular functions are organ morphology and reproductive system development and function. When genes in young rat were compared with those in the aged rat, sperm capacitation related genes were down-regulated in the old rat. However, when genes in the old rat were compared with those in the old rat treated with KRG, KRG treatment up-regulated C21-steroid hormone metabolism. Taken together, Cyp11a1 expression is decreased in the aged rat, however, it is up-regulated by KRG suggesting that KRG seems enhance testes function via Cyp11a1. PMID:23717070

Genome complexity in the coelacanth is reflected in its adaptive immune system

USGS Publications Warehouse

Saha, Nil Ratan; Ota, Tatsuya; Litman, Gary W.; Hansen, John; Parra, Zuly; Hsu, Ellen; Buonocore, Francesco; Canapa, Adriana; Cheng, Jan-Fang; Amemiya, Chris T.

2014-01-01

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

PubMed

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-05-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme▿

PubMed Central

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-01-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions. PMID:19270101

Dynamics of place, boundary and object encoding in rat anterior claustrum

PubMed Central

Jankowski, Maciej M.; O’Mara, Shane M.

2015-01-01

Discrete populations of brain cells signal differing types of spatial information. These “spatial cells” are largely confined to a closely-connected network of sites. We describe here, for the first time, cells in the anterior claustrum of the freely-moving rat encoding place, boundary and object information. This novel claustral spatial signal potentially directly modulates a wide variety of anterior cortical regions. We hypothesize that one of the functions of the claustrum is to provide information about body position, boundaries and landmark information, enabling dynamic control of behavior. PMID:26557060

Identifying the integrated neural networks involved in capsaicin-induced pain using fMRI in awake TRPV1 knockout and wild-type rats

PubMed Central

Yee, Jason R.; Kenkel, William; Caccaviello, John C.; Gamber, Kevin; Simmons, Phil; Nedelman, Mark; Kulkarni, Praveen; Ferris, Craig F.

2015-01-01

In the present study, we used functional MRI in awake rats to investigate the pain response that accompanies intradermal injection of capsaicin into the hindpaw. To this end, we used BOLD imaging together with a 3D segmented, annotated rat atlas and computational analysis to identify the integrated neural circuits involved in capsaicin-induced pain. The specificity of the pain response to capsaicin was tested in a transgenic model that contains a biallelic deletion of the gene encoding for the transient receptor potential cation channel subfamily V member 1 (TRPV1). Capsaicin is an exogenous ligand for the TRPV1 receptor, and in wild-type rats, activated the putative pain neural circuit. In addition, capsaicin-treated wild-type rats exhibited activation in brain regions comprising the Papez circuit and habenular system, systems that play important roles in the integration of emotional information, and learning and memory of aversive information, respectively. As expected, capsaicin administration to TRPV1-KO rats failed to elicit the robust BOLD activation pattern observed in wild-type controls. However, the intradermal injection of formalin elicited a significant activation of the putative pain pathway as represented by such areas as the anterior cingulate, somatosensory cortex, parabrachial nucleus, and periaqueductal gray. Notably, comparison of neural responses to capsaicin in wild-type vs. knock-out rats uncovered evidence that capsaicin may function in an antinociceptive capacity independent of TRPV1 signaling. Our data suggest that neuroimaging of pain in awake, conscious animals has the potential to inform the neurobiological basis of full and integrated perceptions of pain. PMID:25745388

MicroRNA expression in rat brain exposed to repeated inescapable shock: differential alterations in learned helplessness vs. non-learned helplessness.

PubMed

Smalheiser, Neil R; Lugli, Giovanni; Rizavi, Hooriyah S; Zhang, Hui; Torvik, Vetle I; Pandey, Ghanshyam N; Davis, John M; Dwivedi, Yogesh

2011-11-01

MicroRNA (miRNA) expression was measured within frontal cortex of male Holtzman rats subjected to repeated inescapable shocks at days 1 and 7, tested for learned helplessness (LH) at days 2 and 8, and sacrificed at day 15. We compared rats that did vs. did not exhibit LH, as well as rats that were placed in the apparatus and tested for avoidance but not given shocks (tested controls, TC). Non-learned helpless (NLH) rats showed a robust adaptive miRNA response to inescapable shock whereas LH rats showed a markedly blunted response. One set of 12 miRNAs showed particularly large, significant down-regulation in NLH rats relative to tested controls (mir-96, 141, 182, 183, 183*, 298, 200a, 200a*, 200b, 200b*, 200c, 429). These were encoded at a few shared polycistronic loci, suggesting that the down-regulation was coordinately controlled at the level of transcription. Most of these miRNAs are enriched in synaptic fractions. Moreover, almost all of these share 5'-seed motifs with other members of the same set, suggesting that they will hit similar or overlapping sets of target mRNAs. Finally, half of this set is predicted to hit Creb1 as a target. We also identified a core miRNA co-expression module consisting of 36 miRNAs that are highly correlated with each other across individuals of the LH group (but not in the NLH or TC groups). Thus, miRNAs participate in the alterations of gene expression networks that underlie the normal (NLH) as well as aberrant (LH) response to repeated shocks.

Amelioration of hypertensive heart failure by amlodipine may occur via antioxidative effects.

PubMed

Hasegawa, Hiroshi; Takano, Hiroyuki; Kohro, Takahide; Ueda, Kazutaka; Niitsuma, Yuriko; Aburatani, Hiroyuki; Komuro, Issei

2006-09-01

Although recent clinical studies have suggested that long-acting calcium channel blockers (CCBs) have beneficial effects on heart failure, the precise mechanism is unknown. In this study, Dahl salt-sensitive rats fed a high salt diet were treated with the long-acting CCB amlodipine, the low-molecular-weight membrane permeable superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol), or saline from 11 weeks after birth. The cardiac geometry and function, and gene expression profiles were determined at 17 weeks. Dahl salt-sensitive rats fed a high salt diet followed by saline as a non-treatment control (HS group) showed a marked increase in blood pressure and developed concentric hypertrophy at 11 weeks, followed by left ventricular (LV) dilation and congestive heart failure by 17 weeks. The treatment with amlodipine (AMLO group) or Tempol (TEMP group) significantly inhibited the development of LV hypertrophy and cardiac dysfunction. Analysis using an Affymetrix GeneChip U34 revealed that the expression levels of 195 genes were changed by the treatment with amlodipine. Among these 195 genes, 110 genes were increased in HS rats and decreased in AMLO rats. And of these 110 genes, 54 genes were also decreased in TEMP rats. In contrast, 85 genes were decreased in HS rats and increased in AMLO rats. Of these 85 genes, 38 genes were also increased in TEMP rats. Approximately 48% of the genes were changed in similar fashion in AMLO and TEMP rats, suggesting that amlodipine shows beneficial effects on heart failure mainly via antioxidative mechanisms.

Forty-four novel protein-coding loci discovered using a proteomics informed by transcriptomics (PIT) approach in rat male germ cells.

PubMed

Chocu, Sophie; Evrard, Bertrand; Lavigne, Régis; Rolland, Antoine D; Aubry, Florence; Jégou, Bernard; Chalmel, Frédéric; Pineau, Charles

2014-11-01

Spermatogenesis is a complex process, dependent upon the successive activation and/or repression of thousands of gene products, and ends with the production of haploid male gametes. RNA sequencing of male germ cells in the rat identified thousands of novel testicular unannotated transcripts (TUTs). Although such RNAs are usually annotated as long noncoding RNAs (lncRNAs), it is possible that some of these TUTs code for protein. To test this possibility, we used a "proteomics informed by transcriptomics" (PIT) strategy combining RNA sequencing data with shotgun proteomics analyses of spermatocytes and spermatids in the rat. Among 3559 TUTs and 506 lncRNAs found in meiotic and postmeiotic germ cells, 44 encoded at least one peptide. We showed that these novel high-confidence protein-coding loci exhibit several genomic features intermediate between those of lncRNAs and mRNAs. We experimentally validated the testicular expression pattern of two of these novel protein-coding gene candidates, both highly conserved in mammals: one for a vesicle-associated membrane protein we named VAMP-9, and the other for an enolase domain-containing protein. This study confirms the potential of PIT approaches for the discovery of protein-coding transcripts initially thought to be untranslated or unknown transcripts. Our results contribute to the understanding of spermatogenesis by characterizing two novel proteins, implicated by their strong expression in germ cells. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD000872. © 2014 by the Society for the Study of Reproduction, Inc.

Androgen Receptor Involvement in Rat Amelogenesis: An Additional Way for Endocrine-Disrupting Chemicals to Affect Enamel Synthesis.

PubMed

Jedeon, Katia; Loiodice, Sophia; Salhi, Khaled; Le Normand, Manon; Houari, Sophia; Chaloyard, Jessica; Berdal, Ariane; Babajko, Sylvie

2016-11-01

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERβ remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

Lentiviral-mediated Targeted NF-κB Blockade in Dorsal Spinal Cord Glia Attenuates Sciatic Nerve Injury-induced Neuropathic Pain in the Rat.

PubMed

Meunier, Alice; Latrémolière, Alban; Dominguez, Elisa; Mauborgne, Annie; Philippe, Stéphanie; Hamon, Michel; Mallet, Jacques; Benoliel, Jean-Jacques; Pohl, Michel

2007-04-01

Neuropathic pain developing after peripheral nerve injury is associated with altered neuronal and glial cell functions in the spinal cord. Activated glia produces algogenic mediators, exacerbating pain. Among the different intracellular pathways possibly involved in the modified glial function, the nuclear factor κB (NF-κB) system is of particular interest, as numerous genes encoding inflammation- and pain-related molecules are controlled by this transcription factor. NF-κB is a pleiotropic factor also involved in central nervous system homeostasy. To study its role in chronic pain, it is thus essential to inhibit the NF-κB pathway selectively in activated spinal glial cells. Here, we show that when restricted to spinal cord and targeted to glial cells, lentiviral vector-mediated delivery of NF-κB super- repressor IκBα resulted in an inhibition of the NF-κB pathway activated in the rat spinal cord after sciatic nerve injury (chronic constriction injury, CCI). Concomitantly, IκBα overproduction prevented the enhanced expression of interleukin-6 and of inducible nitric oxide synthase associated with chronic constriction injury and resulted in prolonged antihyperalgesic and antiallodynic effects. These data show that targeted blockade of NF-κB activity in spinal glia efficiently alleviates pain behavior in CCI rats, demonstrating the active participation of the glial NF-κB pathway in the development of neuropathic pain after peripheral nerve injury. Copyright © 2007 The American Society of Gene Therapy. Published by Elsevier Inc. All rights reserved.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Arbitrary associations in animals: what can paired associate recall in rats tell us about the neural basis of episodic memory? Theoretical comment on Kesner, Hunsaker, & Warthen (2008).

PubMed

Langston, Rosamund F; Wood, Emma R

2008-12-01

Detailed memories for unique episodes from an individual's past can be triggered, often effortlessly, when that individual is exposed to a stimulus that was present during the original event. The aim of Kesner et al. is to understand the neural basis of memory encoding that supports this cued recall of episodic memories. Kesner and colleagues make novel use of an object-place paired-associate task for rats to provide evidence for a critical role of dorsal CA3 in certain aspects of episodic memory encoding. Using one-trial cued recall versions of the task they show that when rats are cued with an object stimulus, they can be trained to revisit the location in which the object appeared previously. Conversely, when rats are cued with a location, they can learn to choose the object with which it was associated. Rats with dorsal CA3 lesions are severely impaired at these tasks. These data are consistent with the theory that the autoassociative network in CA3 supports the rapid formation of novel associations and may allow pattern completion--the phenomenom whereby a subset of the cues present at an encoding event triggers recall of the whole event. Although flexible recall of arbitrary associations is not fully demonstrated, the study contributes 2 novel behavioral tasks to the previously limited repertoire for studying paired associate recall in rats. It also builds on previous data to specify the role of the hippocampal CA3 subregion in cued recall--a critical aspect of episodic memory.

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonalmore » monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.« less

Transcriptional responses in thyroid tissues from rats treated with a tumorigenic and a non-tumorigenic triazole conazole fungicide.

PubMed

Hester, Susan D; Nesnow, Stephen

2008-03-15

Conazoles are azole-containing fungicides that are used in agriculture and medicine. Conazoles can induce follicular cell adenomas of the thyroid in rats after chronic bioassay. The goal of this study was to identify pathways and networks of genes that were associated with thyroid tumorigenesis through transcriptional analyses. To this end, we compared transcriptional profiles from tissues of rats treated with a tumorigenic and a non-tumorigenic conazole. Triadimefon, a rat thyroid tumorigen, and myclobutanil, which was not tumorigenic in rats after a 2-year bioassay, were administered in the feed to male Wistar/Han rats for 30 or 90 days similar to the treatment conditions previously used in their chronic bioassays. Thyroid gene expression was determined using high density Affymetrix GeneChips (Rat 230_2). Gene expression was analyzed by the Gene Set Expression Analyses method which clearly separated the tumorigenic treatments (tumorigenic response group (TRG)) from the non-tumorigenic treatments (non-tumorigenic response group (NRG)). Core genes from these gene sets were mapped to canonical, metabolic, and GeneGo processes and these processes compared across group and treatment time. Extensive analyses were performed on the 30-day gene sets as they represented the major perturbations. Gene sets in the 30-day TRG group had over representation of fatty acid metabolism, oxidation, and degradation processes (including PPARgamma and CYP involvement), and of cell proliferation responses. Core genes from these gene sets were combined into networks and found to possess signaling interactions. In addition, the core genes in each gene set were compared with genes known to be associated with human thyroid cancer. Among the genes that appeared in both rat and human data sets were: Acaca, Asns, Cebpg, Crem, Ddit3, Gja1, Grn, Jun, Junb, and Vegf. These genes were major contributors in the previously developed network from triadimefon-treated rat thyroids. It is postulated that triadimefon induces oxidative response genes and activates the nuclear receptor, Ppargamma, initiating transcription of gene products and signaling to a series of genes involved in cell proliferation.

Odor Memory and Discrimination Covary as a Function of Delay between Encoding and Recall in Rats.

PubMed

Hackett, Chelsea; Choi, Christina; O'Brien, Brenna; Shin, Philip; Linster, Christiane

2015-06-01

Nonassociative odor learning paradigms are often used to assess memory, social recognition and neuromodulation of olfactory pathways. We here use a modified object recognition paradigm to investigate how an important task parameter, delay between encoding and recall trials, affects the properties of this memory. We show that both memory for a previously investigated odorant and discrimination of a novel odorant decay with delay time and that rats can remember an odorant for up to 45min after a single trial encoding event. The number of odorants that can be encoded, as well as the specificity of the encoded memory, decrease with increased delay and also depend on stimulus concentration. Memory for an odorant and discrimination of a novel odorant decay at approximately the same rate, whereas the specificity of the formed memory decays faster than the memory itself. These results have important implications for the interpretation of behavioral data obtained with this paradigm. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

PubMed Central

Fisher, Susan H.; Wray, Lewis V.

2002-01-01

Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression. PMID:11914346

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

PubMed

Fisher, Susan H; Wray, Lewis V

2002-04-01

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

1994-01-01

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

Analysis of disease-associated objects at the Rat Genome Database

PubMed Central

Wang, Shur-Jen; Laulederkind, Stanley J. F.; Hayman, G. T.; Smith, Jennifer R.; Petri, Victoria; Lowry, Timothy F.; Nigam, Rajni; Dwinell, Melinda R.; Worthey, Elizabeth A.; Munzenmaier, Diane H.; Shimoyama, Mary; Jacob, Howard J.

2013-01-01

The Rat Genome Database (RGD) is the premier resource for genetic, genomic and phenotype data for the laboratory rat, Rattus norvegicus. In addition to organizing biological data from rats, the RGD team focuses on manual curation of gene–disease associations for rat, human and mouse. In this work, we have analyzed disease-associated strains, quantitative trait loci (QTL) and genes from rats. These disease objects form the basis for seven disease portals. Among disease portals, the cardiovascular disease and obesity/metabolic syndrome portals have the highest number of rat strains and QTL. These two portals share 398 rat QTL, and these shared QTL are highly concentrated on rat chromosomes 1 and 2. For disease-associated genes, we performed gene ontology (GO) enrichment analysis across portals using RatMine enrichment widgets. Fifteen GO terms, five from each GO aspect, were selected to profile enrichment patterns of each portal. Of the selected biological process (BP) terms, ‘regulation of programmed cell death’ was the top enriched term across all disease portals except in the obesity/metabolic syndrome portal where ‘lipid metabolic process’ was the most enriched term. ‘Cytosol’ and ‘nucleus’ were common cellular component (CC) annotations for disease genes, but only the cancer portal genes were highly enriched with ‘nucleus’ annotations. Similar enrichment patterns were observed in a parallel analysis using the DAVID functional annotation tool. The relationship between the preselected 15 GO terms and disease terms was examined reciprocally by retrieving rat genes annotated with these preselected terms. The individual GO term–annotated gene list showed enrichment in physiologically related diseases. For example, the ‘regulation of blood pressure’ genes were enriched with cardiovascular disease annotations, and the ‘lipid metabolic process’ genes with obesity annotations. Furthermore, we were able to enhance enrichment of neurological diseases by combining ‘G-protein coupled receptor binding’ annotated genes with ‘protein kinase binding’ annotated genes. Database URL: http://rgd.mcw.edu PMID:23794737

Green tea polyphenols reduce body weight in rats by modulating obesity-related genes.

PubMed

Lu, Chuanwen; Zhu, Wenbin; Shen, Chwan-Li; Gao, Weimin

2012-01-01

Beneficial effects of green tea polyphenols (GTP) against obesity have been reported, however, the mechanism of this protection is not clear. Therefore, the objective of this study was to identify GTP-targeted genes in obesity using the high-fat-diet-induced obese rat model. A total of three groups (n = 12/group) of Sprague Dawley (SD) female rats were tested, including the control group (rats fed with low-fat diet), the HF group (rats fed with high-fat diet), and the HF+GTP group (rats fed with high-fat diet and GTP in drinking water). The HF group increased body weight as compared to the control group. Supplementation of GTP in the drinking water in the HF+GTP group reduced body weight as compared to the HF group. RNA from liver samples was extracted for gene expression analysis. A total of eighty-four genes related to obesity were analyzed using PCR array. Compared to the rats in the control group, the rats in the HF group had the expression levels of 12 genes with significant changes, including 3 orexigenic genes (Agrp, Ghrl, and Nr3c1); 7 anorectic genes (Apoa4, Cntf, Ghr, IL-1β, Ins1, Lepr, and Sort); and 2 genes that relate to energy expenditure (Adcyap1r1 and Adrb1). Intriguingly, the HF+GTP group restored the expression levels of these genes in the high-fat-induced obese rats. The protein expression levels of IL-1β and IL-6 in the serum samples from the control, HF, and HF+GTP groups confirmed the results of gene expression. Furthermore, the protein expression levels of superoxide dismutase-1 (SOD1) and catechol-O-methyltransferase (COMT) also showed GTP-regulated protective changes in this obese rat model. Collectively, this study revealed the beneficial effects of GTP on body weight via regulating obesity-related genes, anti-inflammation, anti-oxidant capacity, and estrogen-related actions in high-fat-induced obese rats.

Green Tea Polyphenols Reduce Body Weight in Rats by Modulating Obesity-Related Genes

PubMed Central

Lu, Chuanwen; Zhu, Wenbin; Shen, Chwan-Li; Gao, Weimin

2012-01-01

Beneficial effects of green tea polyphenols (GTP) against obesity have been reported, however, the mechanism of this protection is not clear. Therefore, the objective of this study was to identify GTP-targeted genes in obesity using the high-fat-diet-induced obese rat model. A total of three groups (n = 12/group) of Sprague Dawley (SD) female rats were tested, including the control group (rats fed with low-fat diet), the HF group (rats fed with high-fat diet), and the HF+GTP group (rats fed with high-fat diet and GTP in drinking water). The HF group increased body weight as compared to the control group. Supplementation of GTP in the drinking water in the HF+GTP group reduced body weight as compared to the HF group. RNA from liver samples was extracted for gene expression analysis. A total of eighty-four genes related to obesity were analyzed using PCR array. Compared to the rats in the control group, the rats in the HF group had the expression levels of 12 genes with significant changes, including 3 orexigenic genes (Agrp, Ghrl, and Nr3c1); 7 anorectic genes (Apoa4, Cntf, Ghr, IL-1β, Ins1, Lepr, and Sort); and 2 genes that relate to energy expenditure (Adcyap1r1 and Adrb1). Intriguingly, the HF+GTP group restored the expression levels of these genes in the high-fat-induced obese rats. The protein expression levels of IL-1β and IL-6 in the serum samples from the control, HF, and HF+GTP groups confirmed the results of gene expression. Furthermore, the protein expression levels of superoxide dismutase-1 (SOD1) and catechol-O-methyltransferase (COMT) also showed GTP-regulated protective changes in this obese rat model. Collectively, this study revealed the beneficial effects of GTP on body weight via regulating obesity-related genes, anti-inflammation, anti-oxidant capacity, and estrogen-related actions in high-fat-induced obese rats. PMID:22715380

A highly divergent gene cluster in honey bees encodes a novel silk family.

PubMed

Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

2006-11-01

The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

NASA Astrophysics Data System (ADS)

Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

2016-06-01

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

PubMed

Hsu, Pei-Jen; Wu, Fang-Ju; Kudo, Masataka; Hsiao, Chih-Lun; Hsueh, Aaron J W; Luo, Ching-Wei

2014-01-01

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

1994-10-18

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

Structure, Function, Interaction, Co-evolution of Rice Blast Resistance Genes

USDA-ARS?s Scientific Manuscript database

Rice blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive rice diseases worldwide. Resistance (R) genes to blast encode proteins that detect pathogen signaling molecules encoded by M. oryzae avirulence (AVR) genes. R genes can be a single or a member of clu...

Harnessing pain heterogeneity and RNA transcriptome to identify blood–based pain biomarkers: a novel correlational study design and bioinformatics approach in a graded chronic constriction injury model

PubMed Central

Grace, Peter M.; Hurley, Daniel; Barratt, Daniel T.; Tsykin, Anna; Watkins, Linda R.; Rolan, Paul E.; Hutchinson, Mark R.

2017-01-01

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables. PMID:22697386

Identification of the neurofibromatosis type 1 gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gutmann, D.H.; Wood, D.L.; Collins, F.S.

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to themore » synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.« less

Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system.

PubMed Central

Watson, K L; Konrad, K D; Woods, D F; Bryant, P J

1992-01-01

The tumor suppressor gene lethal(1)aberrant immune response 8 (air8) of Drosophila melanogaster encodes a homolog of the human S6 ribosomal protein. P element insertions that prevent expression of this gene cause overgrowth of the lymph glands (the hematopoietic organs), abnormal blood cell differentiation, and melanotic tumor formation. They also cause delayed development, inhibit growth of most of the larval organs, and lead to larval lethality. Mitotic recombination experiments indicate that the normal S6 gene is required for clone survival in the germ line and imaginal discs. The S6 gene produces a 1.1-kilobase transcript that is abundant throughout development in wild-type animals and in revertants derived from the insertional mutants but is barely detectable in the mutant larvae. cDNAs corresponding to this transcript show a 248-amino acid open reading frame with 75.4% identity and 94.8% similarity to both human and rat S6 ribosomal protein sequences. The results reveal a regulatory function of this ribosomal protein in the hematopoietic system of Drosophila that may be related to its developmentally regulated phosphorylation. Images PMID:1454811

Identification of a differentially-expressed gene in fatty liver of overfeeding geese.

PubMed

Zhao, Ayong; Tang, Huachun; Lu, Sufang; He, Ruiguo

2007-09-01

In response to overfeeding, geese develop fatty liver. To understand the fattening mechanism, mRNA differential display reverse transcription PCR was used to study the gene expression differences between French Landes grey geese and Xupu white geese in conditions of overfeeding and normal feeding. One gene was found to be up-regulated in the fatty liver in both breeds, and it has a 1797 bp cDNA with 83% identity to chicken SELENBP1. The sequence analysis revealed that its open reading frame of 1413 bp encodes a protein of 471 amino acids, which contains a putative conserved domain of 56 kDa selenium binding protein with high homology to its homologues of chicken (95%), rat (86%), mouse (84%), human (86%), monkey (86%), dog (86%), and cattle (86%). The function of this protein has been briefly reviewed based on published information. In tissue expression analysis, the expression of geese SELENBP1 mRNA was found to be higher in liver or kidney than in other tested tissues. The results showed that overfeeding could increase the mRNA expression level of geese SELENBP1.

Molecular genetics of Erwinia amylovora involved in the development of fire blight.

PubMed

Oh, Chang-Sik; Beer, Steven V

2005-12-15

The bacterial plant pathogen, Erwinia amylovora, causes the devastating disease known as fire blight in some Rosaceous plants like apple, pear, quince, raspberry and several ornamentals. Knowledge of the factors affecting the development of fire blight has mushroomed in the last quarter century. On the molecular level, genes encoding a Hrp type III secretion system, genes encoding enzymes involved in synthesis of extracellular polysaccharides and genes facilitating the growth of E. amylovora in its host plants have been characterized. The Hrp pathogenicity island, delimited by genes suggesting horizontal gene transfer, is composed of four distinct regions, the hrp/hrc region, the HEE (Hrp effectors and elicitors) region, the HAE (Hrp-associated enzymes) region, and the IT (Island transfer) region. The Hrp pathogenicity island encodes a Hrp type III secretion system (TTSS), which delivers several proteins from bacteria to plant apoplasts or cytoplasm. E. amylovora produces two exopolysaccharides, amylovoran and levan, which cause the characteristic fire blight wilting symptom in host plants. In addition, other genes, and their encoded proteins, have been characterized as virulence factors of E. amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic activity and iron harvesting. This review summarizes our understanding of the genes and gene products of E. amylovora that are involved in the development of the fire blight disease.

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

PubMed

Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

2007-02-01

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

PubMed Central

Zhou, Hongxia; Huang, Cao; Yang, Min; Landel, Carlisle P; Xia, Pedro Yuxing; Liu, Yong-Jian; Xia, Xu Gang

2009-01-01

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases. PMID:19214245

Myocardial regeneration in adriamycin cardiomyopathy by nuclear expression of GLP1 using ultrasound targeted microbubble destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Shuyuan; Chen, Jiaxi; Huang, Pintong

Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. Methods and results: We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocationmore » of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. Conclusions: Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration. - Highlights: • The activation of nuclear FoxO1 in cardiac muscle cells associated with adriamycin cardiomyopathy. • Myocardial nuclear GLP-1 stimulates myocardial regeneration and reverses adriamycin cardiomyopathy. • The process of myocardial regeneration associated with dedifferentiation and proliferation.« less

Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gersuk, V.H.; Rose, T.M.; Todaro, G.J.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-relatedmore » sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.« less

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

PubMed Central

Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

2007-01-01

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

A Comprehensive Analysis of Nuclear-Encoded Mitochondrial Genes in Schizophrenia.

PubMed

Gonçalves, Vanessa F; Cappi, Carolina; Hagen, Christian M; Sequeira, Adolfo; Vawter, Marquis P; Derkach, Andriy; Zai, Clement C; Hedley, Paula L; Bybjerg-Grauholm, Jonas; Pouget, Jennie G; Cuperfain, Ari B; Sullivan, Patrick F; Christiansen, Michael; Kennedy, James L; Sun, Lei

2018-05-01

The genetic risk factors of schizophrenia (SCZ), a severe psychiatric disorder, are not yet fully understood. Multiple lines of evidence suggest that mitochondrial dysfunction may play a role in SCZ, but comprehensive association studies are lacking. We hypothesized that variants in nuclear-encoded mitochondrial genes influence susceptibility to SCZ. We conducted gene-based and gene-set analyses using summary association results from the Psychiatric Genomics Consortium Schizophrenia Phase 2 (PGC-SCZ2) genome-wide association study comprising 35,476 cases and 46,839 control subjects. We applied the MAGMA method to three sets of nuclear-encoded mitochondrial genes: oxidative phosphorylation genes, other nuclear-encoded mitochondrial genes, and genes involved in nucleus-mitochondria crosstalk. Furthermore, we conducted a replication study using the iPSYCH SCZ sample of 2290 cases and 21,621 control subjects. In the PGC-SCZ2 sample, 1186 mitochondrial genes were analyzed, among which 159 had p values < .05 and 19 remained significant after multiple testing correction. A meta-analysis of 818 genes combining the PGC-SCZ2 and iPSYCH samples resulted in 104 nominally significant and nine significant genes, suggesting a polygenic model for the nuclear-encoded mitochondrial genes. Gene-set analysis, however, did not show significant results. In an in silico protein-protein interaction network analysis, 14 mitochondrial genes interacted directly with 158 SCZ risk genes identified in PGC-SCZ2 (permutation p = .02), and aldosterone signaling in epithelial cells and mitochondrial dysfunction pathways appeared to be overrepresented in this network of mitochondrial and SCZ risk genes. This study provides evidence that specific aspects of mitochondrial function may play a role in SCZ, but we did not observe its broad involvement even using a large sample. Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

The ribosomal protein genes and Minute loci of Drosophila melanogaster

PubMed Central

Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

2007-01-01

Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains

PubMed Central

Thelin, Jonas; Schouenborg, Jens

2008-01-01

Background Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR) system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1) if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2) if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR. The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP) in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO2 laser heat pulses. Results While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different. Conclusion Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting. PMID:18495020

Two rat brain staufen isoforms differentially bind RNA.

PubMed

Monshausen, M; Putz, U; Rehbein, M; Schweizer, M; DesGroseillers, L; Kuhl, D; Richter, D; Kindler, S

2001-01-01

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Identifying metabolic enzymes with multiple types of association evidence

PubMed Central

Kharchenko, Peter; Chen, Lifeng; Freund, Yoav; Vitkup, Dennis; Church, George M

2006-01-01

Background Existing large-scale metabolic models of sequenced organisms commonly include enzymatic functions which can not be attributed to any gene in that organism. Existing computational strategies for identifying such missing genes rely primarily on sequence homology to known enzyme-encoding genes. Results We present a novel method for identifying genes encoding for a specific metabolic function based on a local structure of metabolic network and multiple types of functional association evidence, including clustering of genes on the chromosome, similarity of phylogenetic profiles, gene expression, protein fusion events and others. Using E. coli and S. cerevisiae metabolic networks, we illustrate predictive ability of each individual type of association evidence and show that significantly better predictions can be obtained based on the combination of all data. In this way our method is able to predict 60% of enzyme-encoding genes of E. coli metabolism within the top 10 (out of 3551) candidates for their enzymatic function, and as a top candidate within 43% of the cases. Conclusion We illustrate that a combination of genome context and other functional association evidence is effective in predicting genes encoding metabolic enzymes. Our approach does not rely on direct sequence homology to known enzyme-encoding genes, and can be used in conjunction with traditional homology-based metabolic reconstruction methods. The method can also be used to target orphan metabolic activities. PMID:16571130

[High gene conversion frequency between genes encoding 2-deoxyglucose-6-phosphate phosphatase in 3 Saccharomyces species].

PubMed

Piscopo, Sara-Pier; Drouin, Guy

2014-05-01

Gene conversions are nonreciprocal sequence exchanges between genes. They are relatively common in Saccharomyces cerevisiae, but few studies have investigated the evolutionary fate of gene conversions or their functional impacts. Here, we analyze the evolution and impact of gene conversions between the two genes encoding 2-deoxyglucose-6-phosphate phosphatase in S. cerevisiae, Saccharomyces paradoxus and Saccharomyces mikatae. Our results demonstrate that the last half of these genes are subject to gene conversions among these three species. The greater similarity and the greater percentage of GC nucleotides in the converted regions, as well as the absence of long regions of adjacent common converted sites, suggest that these gene conversions are frequent and occur independently in all three species. The high frequency of these conversions probably result from the fact that they have little impact on the protein sequences encoded by these genes.

Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat.

PubMed

Michalkiewicz, Mieczyslaw; Michalkiewicz, Teresa; Ettinger, Ruth A; Rutledge, Elizabeth A; Fuller, Jessica M; Moralejo, Daniel H; Van Yserloo, Brian; MacMurray, Armand J; Kwitek, Anne E; Jacob, Howard J; Lander, Eric S; Lernmark, Ake

2004-10-04

A single point mutation in a novel immune-associated nucleotide gene 5 (Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344.lyp/lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344.lyp/lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.

Cerebellar re-encoding of self-generated head movements

PubMed Central

Dugué, Guillaume P; Tihy, Matthieu; Gourévitch, Boris; Léna, Clément

2017-01-01

Head movements are primarily sensed in a reference frame tied to the head, yet they are used to calculate self-orientation relative to the world. This requires to re-encode head kinematic signals into a reference frame anchored to earth-centered landmarks such as gravity, through computations whose neuronal substrate remains to be determined. Here, we studied the encoding of self-generated head movements in the rat caudal cerebellar vermis, an area essential for graviceptive functions. We found that, contrarily to peripheral vestibular inputs, most Purkinje cells exhibited a mixed sensitivity to head rotational and gravitational information and were differentially modulated by active and passive movements. In a subpopulation of cells, this mixed sensitivity underlay a tuning to rotations about an axis defined relative to gravity. Therefore, we show that the caudal vermis hosts a re-encoded, gravitationally polarized representation of self-generated head kinematics in freely moving rats. DOI: http://dx.doi.org/10.7554/eLife.26179.001 PMID:28608779

Low-dose systemic scopolamine disrupts context conditioning in rats.

PubMed

Luyten, Laura; Nuyts, Shauni; Beckers, Tom

2017-06-01

Cholinergic neurotransmission plays a key role in learning and memory. Prior research with rats indicated that a low dose of pre-training scopolamine (0.1 mg/kg), a cholinergic receptor antagonist, did not affect cued fear conditioning, but did block renewal when injected before extinguishing a conditioned tone, opening up opportunities to pharmacologically improve exposure therapy for anxiety patients. Before translating these findings to the clinic, it is important to carefully examine how scopolamine affects contextual fear memories. Here, we investigated the effects of scopolamine on encoding of contextual anxiety and its generalization in male Wistar rats. We found a profound disruption of context conditioning, suggesting that, even at a low dose, systemic scopolamine may influence contextual encoding in the hippocampus, particularly when the context is the best predictor for the presence of shocks.

RNA sequencing analysis reveals new findings of hyperbaric oxygen treatment on rats with acute carbon monoxide poisoning.

PubMed

Wang, Wenlan; Xue, Li; Li, Ya; Li, Rong; Xie, Xiaoping; Bao, Junxiang; Hai, Chunxu; Li, Jinsheng

2016-01-01

To elucidate the altered gene network in the brains of carbon monoxide (CO) poisoned rats after treatment with hyperbaric oxygen (HBO₂). RNA sequencing (RNA-seq) analysis was performed to examine differentially expressed genes (DEGs) in brain tissue samples from nine male rats: a normal control group; a CO poisoning group; and an HBO₂ treatment group (three rats/group). Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR were used for validation of the DEGs in another 18 male rats (six rats/group). RNA-seq revealed that two genes were upregulated (4.18 and 8.76 log to the base 2 fold change) (p⟨0.05) in the CO-poisoned rats relative to the control rats; two genes were upregulated (3.88 and 7.69 log to the base 2 fold change); and 23 genes were downregulated (3.49-15.12 log to the base 2 fold change) (p⟨0.05) in the brains of the HBO₂-treated rats relative to the CO-poisoned rats. Target prediction of DEGs by gene network analysis and analysis of pathways affected suggested that regulation of gene expressions of dopamine metabolism and nitric oxide (NO) synthesis were significantly affected by CO poisoning and HBO₂ treatment. Results of RT-PCR and real-time quantitative PCR indicated that four genes (Pomc, GH-1, Pr1 and Fshβ) associated with hormone secretion in the hypothalamic-pituitary system have potential as markers for prognosis of CO. This study is the first RNA-seq analysis profile of HBO₂ treatment on rats with acute CO poisoning. It concludes that changes of hormone secretion in the hypothalamic-pituitary system, dopamine metabolism and NO synthesis involved in brain damage and behavior abnormalities after CO poisoning and HBO₂ therapy may regulate these changes.

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Treesearch

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

PubMed

Feng, Rui; Liu, Yan; Sun, Xuefei; Wang, Yan; Hu, Huiyuan; Guo, Feng; Zhao, Jinsheng; Hao, Liying

2015-06-01

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

RatLab: an easy to use tool for place code simulations

PubMed Central

Schönfeld, Fabian; Wiskott, Laurenz

2013-01-01

In this paper we present the RatLab toolkit, a software framework designed to set up and simulate a wide range of studies targeting the encoding of space in rats. It provides open access to our modeling approach to establish place and head direction cells within unknown environments and it offers a set of parameters to allow for the easy construction of a variety of enclosures for a virtual rat as well as controlling its movement pattern over the course of experiments. Once a spatial code is formed RatLab can be used to modify aspects of the enclosure or movement pattern and plot the effect of such modifications on the spatial representation, i.e., place and head direction cell activity. The simulation is based on a hierarchical Slow Feature Analysis (SFA) network that has been shown before to establish a spatial encoding of new environments using visual input data only. RatLab encapsulates such a network, generates the visual training data, and performs all sampling automatically—with each of these stages being further configurable by the user. RatLab was written with the intention to make our SFA model more accessible to the community and to that end features a range of elements to allow for experimentation with the model without the need for specific programming skills. PMID:23908627

Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats.

PubMed

Nasirinezhad, Farinaz; Gajavelli, Shyam; Priddy, Blake; Jergova, Stanislava; Zadina, James; Sagen, Jacqueline

2015-01-07

The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy. Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1-5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in these animals using the SHG-EMs combination vectors compared to the group with early intervention. Findings from this study support the potential for direct gene therapy to provide a robust and sustained alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance development for long-term management of persistent pain.

Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

1995-01-01

Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

The Use of Protein-DNA, Chromatin Immunoprecipitation, and Transcriptome Arrays to Describe Transcriptional Circuits in the Dehydrated Male Rat Hypothalamus

PubMed Central

Qiu, Jing; Kleineidam, Anna; Gouraud, Sabine; Yao, Song Tieng; Greenwood, Mingkwan; Hoe, See Ziau; Hindmarch, Charles

2014-01-01

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase. PMID:25144923

A deep auto-encoder model for gene expression prediction.

PubMed

Xie, Rui; Wen, Jia; Quitadamo, Andrew; Cheng, Jianlin; Shi, Xinghua

2017-11-17

Gene expression is a key intermediate level that genotypes lead to a particular trait. Gene expression is affected by various factors including genotypes of genetic variants. With an aim of delineating the genetic impact on gene expression, we build a deep auto-encoder model to assess how good genetic variants will contribute to gene expression changes. This new deep learning model is a regression-based predictive model based on the MultiLayer Perceptron and Stacked Denoising Auto-encoder (MLP-SAE). The model is trained using a stacked denoising auto-encoder for feature selection and a multilayer perceptron framework for backpropagation. We further improve the model by introducing dropout to prevent overfitting and improve performance. To demonstrate the usage of this model, we apply MLP-SAE to a real genomic datasets with genotypes and gene expression profiles measured in yeast. Our results show that the MLP-SAE model with dropout outperforms other models including Lasso, Random Forests and the MLP-SAE model without dropout. Using the MLP-SAE model with dropout, we show that gene expression quantifications predicted by the model solely based on genotypes, align well with true gene expression patterns. We provide a deep auto-encoder model for predicting gene expression from SNP genotypes. This study demonstrates that deep learning is appropriate for tackling another genomic problem, i.e., building predictive models to understand genotypes' contribution to gene expression. With the emerging availability of richer genomic data, we anticipate that deep learning models play a bigger role in modeling and interpreting genomics.

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

PubMed Central

Schreiter, Eric R.; Hasseman, Jeremy P.; Tsegaye, Getahun; Fosque, Benjamin F.; Behnam, Reza; Shields, Brenda C.; Ramirez, Melissa; Kimmel, Bruce E.; Kerr, Rex A.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.

2013-01-01

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude. PMID:24155972

Sexual dimorphism in obesity-related genes in the epicardial fat during aging.

PubMed

Kocher, Caitlin; Christiansen, Matthew; Martin, Sarah; Adams, Christopher; Wehner, Paulette; Gress, Todd; Santanam, Nalini

2017-05-01

Aging increases the risk of cardiovascular disease and metabolic syndrome. Alterations in epicardial fat play an important pathophysiological role in coronary artery disease and hypertension. We investigated the impact of normal aging on obesity-related genes in epicardial fat. Sex-specific changes in obesity-related genes with aging in epicardial fat (EF) were determined in young (6 months) and old (30/36 months) female and male, Fischer 344 × Brown Norway hybrid (FBN) rats, using a rat obesity RT 2 PCR Array. Circulating sex hormone levels, body and heart weights were determined. Statistical significance was determined using two-tailed Student's t test and Pearson's correlation. Our results revealed sex-specific differences in obesity-related genes with aging. Dramatic changes in the expression profile of obesity-related genes in EF with aging in female, but not in male, FBN rats were observed. The older (30 months) female rats had more significant variations in the abundance of obesity-related genes in the EF compared to that seen in younger female rats or both age groups in male rats. A correlation of changes in obesity-related genes in EF to heart weights was observed in female rats, but not in male rats with aging. No correlation was observed to circulating sex hormone levels. Our findings indicate a dysfunctional EF in female rats with aging compared to male rats. These findings, with further functional validation, might help explain the sex differences in cardiovascular risk and mortality associated with aging observed in humans.

Transcriptome profiling of visceral adipose tissue in a novel obese rat model, WNIN/Ob & its comparison with other animal models.

PubMed

Sakamuri, Siva Sankara Vara Prasad; Putcha, Uday Kumar; Veettil, Giridharan Nappan; Ayyalasomayajula, Vajreswari

2016-09-01

Adipose tissue dysfunction in obesity is linked to the development of type 2 diabetes and cardiovascular diseases. We studied the differential gene expression in retroperitoneal adipose tissue of a novel obese rat model, WNIN/Ob, to understand the possible underlying transcriptional changes involved in the development of obesity and associatedcomorbidities in this model. Four month old, male WNIN/Ob lean and obese rats were taken, blood was collected and tissues were dissected. Body composition analysis and adipose tissue histology were performed. Global gene expression in retroperitoneal adipose tissue of lean and obese rats was studied by microarray using Affymetrix GeneChips. One thousand and seventeen probe sets were downregulated and 963 probe sets were upregulated (more than two-fold) in adipose tissue of WNIN/Ob obese rats when compared to that of lean rats. Small nucleolar RNA (SnoRNA) made most of the underexpressed probe sets, whereas immune system-related genes werethe most overexpressed in the adipose tissues of obese rats. Genes coding for cytoskeletal proteinswere downregulated, whereas genes related to lipid biosynthesis were elevated in the adipose tissue of obese rats. Majority of the altered genes and pathways in adipose tissue of WNIN/Ob obese rats were similar to the observations in other obese animal models and human obesity. Based on these observations, it is proposed that WNIN/Ob obese rat model may be a good model to study the mechanisms involved in the development of obesity and its comorbidities. Downregulation of SnoRNA appears to be a novel feature in this obese rat model.

Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

PubMed

Ghoshroy, Sohini; Robertson, Deborah L

2015-01-01

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.

PubMed

Leroch, Michaela; Mernke, Dennis; Koppenhoefer, Dieter; Schneider, Prisca; Mosbach, Andreas; Doehlemann, Gunther; Hahn, Matthias

2011-05-01

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones

PubMed Central

Glass, Jennifer B.; Kretz, Cecilia B.; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L.; Buck, Kristen N.; Landing, William M.; Morton, Peter L.; Moffett, James W.; Giovannoni, Stephen J.; Vergin, Kevin L.; Stewart, Frank J.

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO3−, NO2−, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu. PMID:26441925

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones.

PubMed

Glass, Jennifer B; Kretz, Cecilia B; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L; Buck, Kristen N; Landing, William M; Morton, Peter L; Moffett, James W; Giovannoni, Stephen J; Vergin, Kevin L; Stewart, Frank J

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO[Formula: see text], NO[Formula: see text], Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules

PubMed Central

McArthur, Sarah D; Pulvermacher, Sarah C; Stauffer, George V

2006-01-01

Background In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. Results In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. Conclusion The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA. PMID:16768793

Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

DTIC Science & Technology

2006-07-01

ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

PubMed

Suter, T M; Viswanathan, V K; Cianciotto, N P

1997-06-01

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

PubMed Central

Suter, T M; Viswanathan, V K; Cianciotto, N P

1997-01-01

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae. PMID:9174205

[Preliminary analysis of retinal gene expression profile of diabetic rat].

PubMed

Mei, Yan; Zhou, Hong-ying; Xiang, Tao; Lu, You-guang; Li, Ai-dong; Tang, En-jie; Yang, Hui-jun

2005-10-01

Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat and comparing the profiles in order to analyze the possible genes related with diabetic retinopathy. The whole retinal transcriptional fragments of non-diabetic rat and 8-week diabetic rat were obtained by restriction fragments differential display-PCR (RFDD-PCR). Bioinformatic analysis of retinal gene expression was performed using soft wares, including Fragment Analysis. After comparison of the expression profiles, the related gene fragments of diabetic retinopathy were initially selected as the target gene of further approach. A total of 3639 significant fragments were obtained. By means of more than 3-fold contrast of fluorescent intensity as the differential expression standard, the authors got 840 differential fragments, accounting for 23.08% of the expressed numbers and including 5 visual related genes, 13 excitatory neruotransmitter genes and 3 inhibitory neurotransmitter genes. At the 8th week, the expression of Rhodopsin kinase, beta-arrestin, Phosducinìrod photoreceptor cGMP-gated channel and Rpe65 as well as iGlu R1-4 were down-regulated. mGluRs and GABA-Rs were all up-regulated, whereas the expression of GlyR was unchanged. These results prompt again that the changes in retinal nervous layer of rat have occurred at an early stage of diabetes. The genes expression pattern of visual related genes and excitatory and inhibitory neurotransmitters in rat diabetic retina have been involved in neuro-dysfunctions of diabetic retina.

Potential Role of Axonal Chemorepellent Slit2 in Modulating Adventitial Inflammation in a Rat Carotid Artery Balloon Injury Model

PubMed Central

Liu, Dong; Xiao, Yan; Subramanian, Romesh R.; Okamoto, Ei-ichi; Wilcox, Josiah N.; Anderson, Leonard; De Leon, Hector

2016-01-01

Leukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo—an axonal chemorepellent system in vertebrate and invertebrate development—is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2 and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes. Adventitial inflammatory cells were counted in immunostained arterial sections. E-selectin, vascular CAM-1 (VCAM-1), and intercellular CAM-1 (ICAM-1) were upregulated 2–3 hr after injury, followed by infiltration of neutrophils and monocytes as evidenced by real-time PCR, in situ hybridization, and immunohistochemistry. Slit2, Slit3, and Robo genes exhibited no expression changes at 3 hr; however, they were markedly upregulated 1 day after angioplasty. ICAM-1 expression was reduced by 50%, and the number of adventitial neutrophils decreased by >75% one day after angioplasty. Slit2 has been shown to be a potent chemorepelent of leukocytes, endothelial cells and smooth muscle cells. Thus, we decided to further investigate the localization of Slit2 in injured vessels. Immunohistochemical stainings revealed the presence of Slit2 within the vessel wall and in the perivascular vasa vasorum of naive and injured arteries. Double immunohistochemical analyses showed that infiltrating monocytes expressed Slit2 in the perivascular and adventitial tissues of injured arteries 1 and 3 days postangioplasty. In addition, recombinant full-length Slit2 and Slit2-N/1118, an N-terminal fragment of Slit2, inhibited stromal cell-derived factor 1 (SDF-1)-mediated migration of circulating rat peripheral blood mononuclear cells. In summary, adventitial activation of CAM genes and neutrophil infiltration preceded upregulation of Slit/Robo genes. Sli2 expression colocalized with infiltrating inflammatory cells in the adventitial layer. This temporospatial association suggests that leukocyte chemorepellent Slit2 may be involved in halting the adventitial accumulation of inflammatory cells in injured vessels. PMID:26841069

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

PubMed

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

2017-09-01

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine.

PubMed

Santos-Llamas, Ana; Monte, Maria J; Marin, Jose J G; Perez, Maria J

2018-03-28

The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause-effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35 days. AZT at a concentration of 1 mg/ml, but not 0.5 mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

PubMed

Tulinská, Jana; Adel-Patient, Karine; Bernard, Hervé; Líšková, Aurélia; Kuricová, Miroslava; Ilavská, Silvia; Horváthová, Mira; Kebis, Anton; Rollerová, Eva; Babincová, Júlia; Aláčová, Radka; Wal, Jean-Michel; Schmidt, Kerstin; Schmidtke, Jörg; Schmidt, Paul; Kohl, Christian; Wilhelm, Ralf; Schiemann, Joachim; Steinberg, Pablo

2018-07-01

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

PubMed

Nicolson, Garth L; Nawa, Akihiro; Toh, Yasushi; Taniguchi, Shigeki; Nishimori, Katsuhiko; Moustafa, Amr

2003-01-01

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

PubMed

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora.

PubMed

Pöggeler, S

2000-06-01

In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from a genomic library of Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy methylated C-terminal cysteine residue. The sequences of the predicted pheromones display strong structural similarity to those encoded by putative pheromones of heterothallic filamentous ascomycetes. Both genes are expressed during the life cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus. Southern-hybridization experiments indicated that ppg1 and ppg2 homologues are also present in other homothallic ascomycetes.

Arxula adeninivorans (Blastobotrys adeninivorans) — A Dimorphic Yeast of Great Biotechnological Potential

NASA Astrophysics Data System (ADS)

Böer, Erik; Steinborn, Gerhard; Florschütz, Kristina; Körner, Martina; Gellissen, Gerd; Kunze, Gotthard

The dimorphic ascomycetous yeast Arxula adeninivorans exhibits some unusual properties. Being a thermo- and halotolerant species it is able to assimilate and ferment many compounds as sole carbon and/or nitrogen source. It utilises n-alkanes and is capable of degrading starch. Due to these unusual biochemical properties A. adeninivorans can be exploited as a gene donor for the production of enzymes with attractive biotechnological characteristics. Examples of A. adeninivorans-derived genes that are overexpressed include the ALIP1 gene encoding a secretory lipase, the AINV encoding invertase, the AXDH encoding xylitol dehydrogenase and the APHY encoding a secretory phosphatase with phytase activity.

Wnt/β-catenin signaling cascade down-regulation following massive small bowel resection in a rat.

PubMed

Sukhotnik, Igor; Roitburt, Alex; Pollak, Yulia; Dorfman, Tatiana; Matter, Ibrahim; Mogilner, Jorge G; Bejar, Jacob; Coran, Arnold G

2014-02-01

Growing evidence suggests that the Wnt/β-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, lineage commitment, and cell survival during normal development and tissue regeneration of the gastrointestinal epithelium. The roles of this signaling cascade in stimulation of cell proliferation after massive small bowel resection are unknown. The purpose of this study was to evaluate the role of Wnt/β-catenin signaling during late stages of intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's digital gene expression analysis was used to determine Wnt/β-catenin signaling gene expression profiling. Twelve Wnt/β-catenin-related genes and β-catenin protein expression were determined using real-time PCR, western blotting and immunohistochemistry. From the total number of 20,000 probes, 20 genes related to Wnt/β-catenin signaling were investigated. From these genes, seven genes were found to be up-regulated and eight genes to be down-regulated in SBS vs. sham animals with a relative change in gene expression level of 20 % or more. From 12 genes determined by real-time PCR, nine genes were down-regulated in SBS rats compared to control animals including target gene c-Myc. SBS rats also showed a significant decrease in β-catenin protein compared to control animals. Two weeks following massive bowel resection in rats, Wnt/β-catenin signaling pathway is inhibited. In addition, it appears that cell differentiation rather than proliferation is most important in the late stages of intestinal adaptation.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Rapid bursts of androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals

PubMed Central

2008-01-01

Background The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) α, β and γ subunits. Further investigation of 14 α-like (Abpa) and 13 β- or γ-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals. Results Here, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa. Conclusion We conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification. PMID:18269759

Rapid bursts of androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals.

PubMed

Laukaitis, Christina M; Heger, Andreas; Blakley, Tyler D; Munclinger, Pavel; Ponting, Chris P; Karn, Robert C

2008-02-12

The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) alpha, beta and gamma subunits. Further investigation of 14 alpha-like (Abpa) and 13 beta- or gamma-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals. Here, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa. We conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification.

Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

PubMed Central

Jackson, Kasey L.; Dayton, Robert D.; Deverman, Benjamin E.; Klein, Ronald L.

2016-01-01

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats. PMID:27867348

Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

PubMed

Jackson, Kasey L; Dayton, Robert D; Deverman, Benjamin E; Klein, Ronald L

2016-01-01

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

Bayesian decoding using unsorted spikes in the rat hippocampus

PubMed Central

Layton, Stuart P.; Chen, Zhe; Wilson, Matthew A.

2013-01-01

A fundamental task in neuroscience is to understand how neural ensembles represent information. Population decoding is a useful tool to extract information from neuronal populations based on the ensemble spiking activity. We propose a novel Bayesian decoding paradigm to decode unsorted spikes in the rat hippocampus. Our approach uses a direct mapping between spike waveform features and covariates of interest and avoids accumulation of spike sorting errors. Our decoding paradigm is nonparametric, encoding model-free for representing stimuli, and extracts information from all available spikes and their waveform features. We apply the proposed Bayesian decoding algorithm to a position reconstruction task for freely behaving rats based on tetrode recordings of rat hippocampal neuronal activity. Our detailed decoding analyses demonstrate that our approach is efficient and better utilizes the available information in the nonsortable hash than the standard sorting-based decoding algorithm. Our approach can be adapted to an online encoding/decoding framework for applications that require real-time decoding, such as brain-machine interfaces. PMID:24089403

Fish protein hydrolysates affect cholesterol metabolism in rats fed non-cholesterol and high-cholesterol diets.

PubMed

Hosomi, Ryota; Fukunaga, Kenji; Arai, Hirofumi; Kanda, Seiji; Nishiyama, Toshimasa; Yoshida, Munehiro

2012-03-01

Fish consumption is well known to provide health benefits in both experimental animals and human subjects. Numerous studies have demonstrated the beneficial effects of various protein hydrolysates on lipid metabolism. In this context, this study examined the effect of fish protein hydrolysates (FPH) on cholesterol metabolism compared with the effect of casein. FPHs were prepared from Alaska pollock meat using papain as a protease. Male Wistar rats were divided into the following four dietary groups of seven rats each: either casein (20%) or FPH (10%) + casein (10%), with or without 0.5% cholesterol and 0.1% sodium cholate. Serum and liver lipid levels, fecal cholesterol and bile acid excretions, and the hepatic expression of genes encoding proteins involved in cholesterol homeostasis were examined. In rats fed the FPH diets compared with casein diets with or without cholesterol and sodium cholate, the indexes of cholesterol metabolism-namely, serum cholesterol, triglyceride, and low-density lipoprotein-cholesterol levels-were significantly lower, whereas fecal cholesterol and bile acid excretions were higher. Rats fed the FPH diets compared with casein with cholesterol exhibited a lower liver cholesterol level via an increased liver cholesterol 7α-hydroxylase (CYP7A1) expression level. This study demonstrates that the intake of FPH has hypocholesterolemic effects through the enhancement of fecal cholesterol and bile acid excretions and CYP7A1 expression levels. Therefore, fish peptides prepared by papain digestion might provide health benefits by decreasing the cholesterol content in the blood, which would contribute to the prevention of circulatory system diseases such as arteriosclerosis.

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds.

PubMed

Seabra, Ana R; Vieira, Cristina P; Cullimore, Julie V; Carvalho, Helena G

2010-08-19

Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

PubMed

Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

2011-04-01

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

PubMed Central

Wilbrink, Maarten H.; Casabon, Israël; Stewart, Gordon R.; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D.

2012-01-01

Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. PMID:23024343

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

PubMed

Daniel, Guillaume; Musso, Alessandra; Tsika, Elpida; Fiser, Aris; Glauser, Liliane; Pletnikova, Olga; Schneider, Bernard L; Moore, Darren J

2015-01-01

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato.

PubMed

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Yamaguchi, Tomoyuki; Tamura, Chihiro; Sanbo, Makoto; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2012-11-01

Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.

Bloodmeal Identification in Field-Collected Sand Flies From Casa Branca, Brazil, Using the Cytochrome b PCR Method.

PubMed

Carvalho, G M L; Rêgo, F D; Tanure, A; Silva, A C P; Dias, T A; Paz, G F; Andrade Filho, J D

2017-07-01

PCR-based identification of vertebrate host bloodmeals has been performed on several vectors species with success. In the present study, we used a previously published PCR protocol followed by DNA sequencing based on primers designed from multiple alignments of the mitochondrial cytochrome b gene used to identify avian and mammalian hosts of various hematophagous vectors. The amplification of a fragment encoding a 359 bp sequence of the Cyt b gene yielded recognized amplification products in 192 female sand flies (53%), from a total of 362 females analyzed. In the study area of Casa Branca, Brazil, blood-engorged female sand flies such as Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1924), and Nyssomyia whitmani (Antunes & Coutinho, 1939) were analyzed for bloodmeal sources. The PCR-based method identified human, dog, chicken, and domestic rat blood sources. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

PubMed Central

Reinhart, Alexandria A.; Powell, Daniel A.; Nguyen, Angela T.; O'Neill, Maura; Djapgne, Louise; Wilks, Angela; Ernst, Robert K.

2014-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. PMID:25510881

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

PubMed

Kirilyuk, Alexander; Tolstonog, Genrich V; Damert, Annette; Held, Ulrike; Hahn, Silvia; Löwer, Roswitha; Buschmann, Christian; Horn, Axel V; Traub, Peter; Schumann, Gerald G

2008-02-01

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Low-dose systemic scopolamine disrupts context conditioning in rats

PubMed Central

Luyten, Laura; Nuyts, Shauni; Beckers, Tom

2017-01-01

Cholinergic neurotransmission plays a key role in learning and memory. Prior research with rats indicated that a low dose of pre-training scopolamine (0.1 mg/kg), a cholinergic receptor antagonist, did not affect cued fear conditioning, but did block renewal when injected before extinguishing a conditioned tone, opening up opportunities to pharmacologically improve exposure therapy for anxiety patients. Before translating these findings to the clinic, it is important to carefully examine how scopolamine affects contextual fear memories. Here, we investigated the effects of scopolamine on encoding of contextual anxiety and its generalization in male Wistar rats. We found a profound disruption of context conditioning, suggesting that, even at a low dose, systemic scopolamine may influence contextual encoding in the hippocampus, particularly when the context is the best predictor for the presence of shocks. PMID:28417664

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Paralogous ALT1 and ALT2 Retention and Diversification Have Generated Catalytically Active and Inactive Aminotransferases in Saccharomyces cerevisiae

PubMed Central

Peñalosa-Ruiz, Georgina; Aranda, Cristina; Ongay-Larios, Laura; Colon, Maritrini; Quezada, Hector; Gonzalez, Alicia

2012-01-01

Background Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. Principal Findings Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor. PMID:23049841

Relating genes to function: identifying enriched transcription factors using the ENCODE ChIP-Seq significance tool.

PubMed

Auerbach, Raymond K; Chen, Bin; Butte, Atul J

2013-08-01

Biological analysis has shifted from identifying genes and transcripts to mapping these genes and transcripts to biological functions. The ENCODE Project has generated hundreds of ChIP-Seq experiments spanning multiple transcription factors and cell lines for public use, but tools for a biomedical scientist to analyze these data are either non-existent or tailored to narrow biological questions. We present the ENCODE ChIP-Seq Significance Tool, a flexible web application leveraging public ENCODE data to identify enriched transcription factors in a gene or transcript list for comparative analyses. The ENCODE ChIP-Seq Significance Tool is written in JavaScript on the client side and has been tested on Google Chrome, Apple Safari and Mozilla Firefox browsers. Server-side scripts are written in PHP and leverage R and a MySQL database. The tool is available at http://encodeqt.stanford.edu. abutte@stanford.edu Supplementary material is available at Bioinformatics online.

Progesterone up-regulates vasodilator effects of calcitonin gene-related peptide in N(G)-nitro-L-arginine methyl ester-induced hypertension.

PubMed

Gangula, P R; Wimalawansa, S J; Yallampalli, C

1997-04-01

We recently reported that calcitonin gene-related peptide can reverse the hypertension produced by N(G)-nitro-L-arginine methyl ester in pregnant rats. In the current study we investigated whether these vasodilator effects of calcitonin gene-related peptide were progesterone dependent. Calcitonin gene-related peptide or N(G)-nitro-L-arginine methyl ester was infused through osmotic minipumps, either separately or in combination, to groups of five pregnant rats from day 17 of gestation until day 8 post partum or to nonpregnant ovariectomized rats for 8 days. Progesterone was injected during days 1 to 6 post partum and for 6 days after ovariectomy. Systolic blood pressure was measured daily. Animals receiving N(G)-nitro-L-arginine methyl ester exhibited significant elevations of blood pressure during pregnancy and post partum. Coadministration of calcitonin gene-related peptide to these rats reversed the hypertension during pregnancy but not during the postpartum period. At the dose used in this study calcitonin gene-related peptide administered alone was without significant effects on blood pressure. However, it reduced both the mortality and growth restriction of the fetus associated with N(G)-nitro-L-arginine methyl ester in these animals. Calcitonin gene-related peptide reversed the hypertension in N(G)-nitro-L-arginine methyl ester-infused postpartum rats during the periods of progesterone treatment only, and these effects were lost when progesterone treatment was stopped. Neither progesterone nor calcitonin gene-related peptide alone were effective. To further confirm these observations, progesterone effects were tested in ovariectomized adult rats. Similar to the findings in postpartum rats, calcitonin gene-related peptide completely reversed the elevation in blood pressure in N(G)-nitro-L-arginine methyl ester-treated rats receiving progesterone injections. The effects of calcitonin gene-related peptide were apparent only during the progesterone treatment period, and these effects were lost when progesterone treatment was stopped. Again, at these doses calcitonin gene-related peptide and progesterone were each ineffective alone. Calcitonin gene-related peptide reverses the N(G)-nitro-L-arginine methyl ester-induced hypertension during pregnancy, when progesterone levels are elevated, but not post partum or in ovariectomized nonpregnant rats. The blood pressure-lowering effects of calcitonin gene-related peptide were restored in both postpartum and ovariectomized rats with progesterone treatment. Therefore we conclude that progesterone modulates vasodilator effects of calcitonin gene-related peptide in hypertensive rats.

Gender-related difference in altered gene expression of a sterol regulatory element binding protein, SREBP-2, by lead nitrate in rats: correlation with development of hypercholesterolemia.

PubMed

Kojima, Misaki; Degawa, Masakuni

2006-01-01

Changes in gene expression levels of hepatic sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) after a single i.v. injection of lead nitrate (LN, 100 micromol kg(-1) body weight) were examined comparatively by real time reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats. Significant increases in the gene expression level of SREBP-2, a transcription factor for the HMGR gene, occurred at 6-12 h in male and at 24-36 h in female rats after LN-treatment. The gene expression level of HMGR, a rate-limiting enzyme for cholesterol biosynthesis, significantly increased at 3-48 h in male rats and 12-48 h in female rats. Subsequently, significant increases in the amount of hepatic total cholesterol in male and female rats were also observed at 3-48 h and 24-48 h, respectively. The present findings demonstrate that increases in gene expressions of hepatic SREBP-2 and HMGR and the amount of hepatic total cholesterol by LN occur earlier in male rats than in the females, and that increases in the gene expression level of HMGR and the amount of hepatic total cholesterol occur prior to the increase in the gene expression level of SREBP-2 in either sex of rats. Copyright (c) 2006 John Wiley & Sons, Ltd.

Identification and validation of midbrain Kcnq4 regulation of heavy alcohol consumption in rodents.

PubMed

McGuier, Natalie S; Rinker, Jennifer A; Cannady, Reginald; Fulmer, Diana B; Jones, Sara R; Hoffman, Michaela; Mulholland, Patrick J

2018-05-24

Currently available pharmacotherapies for treating alcohol use disorder (AUD) suffer from deleterious side effects and are not efficacious in diverse populations. Clinical and preclinical studies provide evidence that the Kcnq family of genes that encode K V 7 channels influence alcohol intake and dependence. K V 7 channels are a class of slowly activating voltage-dependent K + channels that regulate neuronal excitability. Studies indicate that the K V 7 channel positive modulator retigabine can decrease dopaminergic neuron firing, alter dopamine (DA) release, and reduce alcohol intake in heavy drinking rodents. Given the critical nature of ventral tegmental area (VTA) DA to the addiction process and predominant expression of Kcnq4 in DA neurons, we investigated the role of midbrain Kcnq genes and K V 7 channels in the VTA of genetically diverse mice and long-term heavy drinking rats, respectively. Integrative bioinformatics analysis identified negative correlations between midbrain Kcnq4 expression and alcohol intake and seeking behaviors. Kcnq4 expression levels were also correlated with dopaminergic-related phenotypes in BXD strains, and Kcnq4 was present in support intervals for alcohol sensitivity and alcohol withdrawal severity QTLs in rodents. Pharmacological validation studies revealed that VTA K V 7 channels regulate excessive alcohol intake in rats with a high-drinking phenotype. Administration of a novel and selective K V 7.2/4 channel positive modulator also reduced alcohol drinking in rats. Together, these findings indicate that midbrain Kcnq4 expression regulates alcohol-related behaviors in genetically diverse mice and provide evidence that K V 7.4 channels are a critical mediator of excessive alcohol drinking. Copyright © 2018 Elsevier Ltd. All rights reserved.

A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells.

PubMed

Guo, Deng-Fu; Tardif, Valerie; Ghelima, Karin; Chan, John S D; Ingelfinger, Julie R; Chen, XiangMei; Chenier, Isabelle

2004-05-14

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.

Rudimentary expression of RYamide in Drosophila melanogaster relative to other Drosophila species points to a functional decline of this neuropeptide gene.

PubMed

Veenstra, Jan A; Khammassi, Hela

2017-04-01

RYamides are arthropod neuropeptides with unknown function. In 2011 two RYamides were isolated from D. melanogaster as the ligands for the G-protein coupled receptor CG5811. The D. melanogaster gene encoding these neuropeptides is highly unusual, as there are four RYamide encoding exons in the current genome assembly, but an exon encoding a signal peptide is absent. Comparing the D. melanogaster gene structure with those from other species, including D. virilis, suggests that the gene is degenerating. RNAseq data from 1634 short sequence read archives at NCBI containing more than 34 billion spots yielded numerous individual spots that correspond to the RYamide encoding exons, of which a large number include the intron-exon boundary at the start of this exon. Although 72 different sequences have been spliced onto this RYamide encoding exon, none codes for the signal peptide of this gene. Thus, the RNAseq data for this gene reveal only noise and no signal. The very small quantities of peptide recovered during isolation and the absence of credible RNAseq data, indicates that the gene is very little expressed, while the RYamide gene structure in D. melanogaster suggests that it might be evolving into a pseudogene. Yet, the identification of the peptides it encodes clearly shows it is still functional. Using region specific antisera, we could localize numerous neurons and enteroendocrine cells in D. willistoni, D. virilis and D. pseudoobscura, but only two adult abdominal neurons in D. melanogaster. Those two neurons project to and innervate the rectal papillae, suggesting that RYamides may be involved in the regulation of water homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus▿

PubMed Central

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E.; Brautaset, Trygve

2011-01-01

We investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for l-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased l-lysine production levels. PMID:21724876

Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus.

PubMed

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E; Brautaset, Trygve

2011-09-01

We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.

Perineural Dexmedetomidine Attenuates Inflammation in Rat Sciatic Nerve via the NF-κB Pathway

PubMed Central

Huang, Yan; Lu, Yi; Zhang, Lei; Yan, Jia; Jiang, Jue; Jiang, Hong

2014-01-01

Recent studies have shown that dexmedetomidine exerts an anti-inflammatory effect by reducing serum levels of inflammatory factors, however, the up-stream mechanism is still unknown. The transcription factor NF-κB enters the nucleus and promotes the transcription of its target genes, including those encoding the pro-inflammatory cytokines IL-6 and TNF-α. In this study, we established a rat model that simulates a clinical surgical procedure to investigate the anti-inflammatory effect of perineural administration of dexmedetomidine and the underlying mechanism. Dexmedetomidine reduced the sciatic nerve levels of IL-6 and TNF-α at both the mRNA and protein level. Dexmedetomidine also inhibited the translocation of activated NF-κB to the nucleus and the binding activity of NF-κB. The anti-inflammatory effect is confirmed to be dose-dependent. Finally, pyrrolidine dithiocarbamate also reduced the levels of IL-6 and TNF-α and the activation of NF-κB. In conclusion, dexmedetomidine inhibited the nuclear translocation and binding activity of activated NF-κB, thus reducing inflammatory cytokines. PMID:24663080

Dynamic Encoding of Incentive Salience in the Ventral Pallidum: Dependence on the Form of the Reward Cue.

PubMed

Ahrens, Allison M; Ferguson, Lindsay M; Robinson, Terry E; Aldridge, J Wayne

2018-01-01

Some rats are especially prone to attribute incentive salience to a cue (conditioned stimulus, CS) paired with food reward (sign-trackers, STs), but the extent they do so varies as a function of the form of the CS. Other rats respond primarily to the predictive value of a cue (goal-trackers, GTs), regardless of its form. Sign-tracking is associated with greater cue-induced activation of mesolimbic structures than goal-tracking; however, it is unclear how the form of the CS itself influences activity in neural systems involved in incentive salience attribution. Thus, our goal was to determine how different cue modalities affect neural activity in the ventral pallidum (VP), which is known to encode incentive salience attribution, as rats performed a two-CS Pavlovian conditioned approach task in which both a lever-CS and a tone-CS predicted identical food reward. The lever-CS elicited sign-tracking in some rats (STs) and goal-tracking in others (GTs), whereas the tone-CS elicited only goal-tracking in all rats. The lever-CS elicited robust changes in neural activity (sustained tonic increases or decreases in firing) throughout the VP in STs, relative to GTs. These changes were not seen when STs were exposed to the tone-CS, and in GTs there were no differences in firing between the lever-CS and tone-CS. We conclude that neural activity throughout the VP encodes incentive signals and is especially responsive when a cue is of a form that promotes the attribution of incentive salience to it, especially in predisposed individuals.

“Guilt by Association” Is the Exception Rather Than the Rule in Gene Networks

PubMed Central

Gillis, Jesse; Pavlidis, Paul

2012-01-01

Gene networks are commonly interpreted as encoding functional information in their connections. An extensively validated principle called guilt by association states that genes which are associated or interacting are more likely to share function. Guilt by association provides the central top-down principle for analyzing gene networks in functional terms or assessing their quality in encoding functional information. In this work, we show that functional information within gene networks is typically concentrated in only a very few interactions whose properties cannot be reliably related to the rest of the network. In effect, the apparent encoding of function within networks has been largely driven by outliers whose behaviour cannot even be generalized to individual genes, let alone to the network at large. While experimentalist-driven analysis of interactions may use prior expert knowledge to focus on the small fraction of critically important data, large-scale computational analyses have typically assumed that high-performance cross-validation in a network is due to a generalizable encoding of function. Because we find that gene function is not systemically encoded in networks, but dependent on specific and critical interactions, we conclude it is necessary to focus on the details of how networks encode function and what information computational analyses use to extract functional meaning. We explore a number of consequences of this and find that network structure itself provides clues as to which connections are critical and that systemic properties, such as scale-free-like behaviour, do not map onto the functional connectivity within networks. PMID:22479173

Effect of Korean Red Ginseng treatment on the gene expression profile of diabetic rat retina

PubMed Central

Yang, Hana; Son, Gun Woo; Park, Hye Rim; Lee, Seung Eun; Park, Yong Seek

2015-01-01

Background Korean Red Ginseng (KRG) is a herbal medicine used in Asian countries and is very popular for its beneficial biological properties. Diabetes mellitus (DM) and its complications are rapidly becoming a global public health concern. The literature on transcriptional changes induced by KRG in rat models of diabetic retinopathy is limited. Considering these facts, we designed this study to determine whether retinopathy-associated genes are altered in retinas of rats with DM and whether the induced changes are reversed by KRG. Methods Male Sprague–Dawley rats were intravenously injected with streptozotocin (50 mg/kg body weight) to induce DM, following which, KRG powder (200 mg/kg body weight) was orally administered to the KRG-treated DM rat group for 10 wks. The rats were then sacrificed, and their retinas were harvested for total RNA extraction. Microarray gene expression profiling was performed on the extracted RNA samples. Results From among > 31,000 genes investigated, the expression of 268 genes was observed to be upregulated and that of 58 genes was downregulated, with twofold altered expression levels in the DM group compared with those in the control group. Moreover, 39 genes were upregulated more than twofold and 84 genes were downregulated in the KRG-treated group compared to the DM group. The expression of the genes was significantly reversed by KRG treatment; some of these genes were analyzed further to verify the results of the microarray experiments. Conclusion Taken together, our data suggest that reversed changes in the gene expression may mediate alleviating activities of KRG in rats with diabetic retinopathy. PMID:26843816

Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

PubMed

Xiao, Xiaolin; Moreno-Moral, Aida; Rotival, Maxime; Bottolo, Leonardo; Petretto, Enrico

2014-01-01

Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed heat shock protein (Hsp) and cardiomyopathy genes (Bag3, Cryab, Kras, Emd, Plec), which was significantly replicated using separate failing heart and liver gene expression datasets in humans, thus revealing a conserved functional role for Hsp genes in cardiovascular disease.

Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.

PubMed

Queck, Shu Y; Khan, Burhan A; Wang, Rong; Bach, Thanh-Huy L; Kretschmer, Dorothee; Chen, Liang; Kreiswirth, Barry N; Peschel, Andreas; Deleo, Frank R; Otto, Michael

2009-07-01

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

PubMed

Trejter, Marcin; Jopek, Karol; Celichowski, Piotr; Tyczewska, Marianna; Malendowicz, Ludwik K; Rucinski, Marcin

2015-01-01

Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRβ were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRβ receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Lecithin Prevents Cortical Cytoskeleton Reorganization in Rat Soleus Muscle Fibers under Short-Term Gravitational Disuse

PubMed Central

Biryukov, Nikolay S.

2016-01-01

The aim of this study was to prevent the cortical cytoskeleton reorganization of rat soleus muscle fibers under short-term gravitational disuse. Once a day, we injected the right soleus muscle with 0.5 ml lecithin at a concentration of 200 mg/ml and the left soleus muscle with a diluted solution in an equal volume for 3 days prior to the experiment. To simulate microgravity conditions in rats, an anti-orthostatic suspension was used according to the Ilyin-Novikov method modified by Morey-Holton et al. for 6 hours. The following groups of soleus muscle tissues were examined: «C», «C+L», «HS», and «HS+L». The transversal stiffness of rat soleus muscle fibers after 6 hours of suspension did not differ from that of the control group for the corresponding legs; there were no differences between the groups without lecithin «C» and «HS» or between the groups with lecithin «C+L» and «HS+L». However, lecithin treatment for three days resulted in an increase in cell stiffness; in the «C+L» group, cell stiffness was significantly higher by 22.7% (p < 0.05) compared with that of group «C». The mRNA content of genes encoding beta- and gamma-actin and beta-tubulin did not significantly differ before and after suspension in the corresponding groups. However, there was a significant increase in the mRNA content of these genes after lecithin treatment: the beta-actin and gamma-actin mRNA content in group «C+L» increased by 200% compared with that of group «C», and beta-tubulin increased by 100% (as well as the mRNA content of tubulin-binding proteins Ckap5, Tcp1, Cct5 and Cct7). In addition, desmin mRNA content remained unchanged in all of the experimental groups. As a result of the lecithin injections, there was a redistribution of the mRNA content of genes encoding actin monomer- and filament-binding proteins in the direction of increasing actin polymerization and filament stability; the mRNA content of Arpc3 and Lcp1 increased by 3- and 5-fold, respectively, but the levels of Tmod1 and Svil decreased by 2- and 5-fold, respectively. However, gravitational disuse did not result in changes in the mRNA content of Arpc3, Tmod1, Svil or Lcp1. Anti-orthostatic suspension for 6 hours resulted in a decrease in the mRNA content of alpha-actinin-4 (Actn4) and alpha-actinin-1 (Actn1) in group «HS» compared with that of group «C» by 25% and 30%, respectively, as well as a decrease and increase in the ACTN4 protein content in the membrane and cytoplasmic fractions, respectively. Lecithin injection resulted in an increase in the Actn1 and Actn4 mRNA content in group «C+L» by 1.5-fold and more than 2-fold, respectively, compared with the levels in group «C». Moreover, in group «HS+L», the mRNA content did not change in these genes compared with the levels in group «C+L», and the ACTN4 protein content in the membrane and cytoplasmic fractions also remained unchanged. Thus, lecithin prevented the reduction of Actn1 and Actn4 mRNA and the migration of ACTN4 from the cortical cytoskeleton to the cytoplasm. PMID:27073851

Lecithin Prevents Cortical Cytoskeleton Reorganization in Rat Soleus Muscle Fibers under Short-Term Gravitational Disuse.

PubMed

Ogneva, Irina V; Biryukov, Nikolay S

2016-01-01

The aim of this study was to prevent the cortical cytoskeleton reorganization of rat soleus muscle fibers under short-term gravitational disuse. Once a day, we injected the right soleus muscle with 0.5 ml lecithin at a concentration of 200 mg/ml and the left soleus muscle with a diluted solution in an equal volume for 3 days prior to the experiment. To simulate microgravity conditions in rats, an anti-orthostatic suspension was used according to the Ilyin-Novikov method modified by Morey-Holton et al. for 6 hours. The following groups of soleus muscle tissues were examined: "C", "C+L", "HS", and "HS+L". The transversal stiffness of rat soleus muscle fibers after 6 hours of suspension did not differ from that of the control group for the corresponding legs; there were no differences between the groups without lecithin «C» and «HS» or between the groups with lecithin "C+L" and "HS+L". However, lecithin treatment for three days resulted in an increase in cell stiffness; in the "C+L" group, cell stiffness was significantly higher by 22.7% (p < 0.05) compared with that of group "C". The mRNA content of genes encoding beta- and gamma-actin and beta-tubulin did not significantly differ before and after suspension in the corresponding groups. However, there was a significant increase in the mRNA content of these genes after lecithin treatment: the beta-actin and gamma-actin mRNA content in group "C+L" increased by 200% compared with that of group "C", and beta-tubulin increased by 100% (as well as the mRNA content of tubulin-binding proteins Ckap5, Tcp1, Cct5 and Cct7). In addition, desmin mRNA content remained unchanged in all of the experimental groups. As a result of the lecithin injections, there was a redistribution of the mRNA content of genes encoding actin monomer- and filament-binding proteins in the direction of increasing actin polymerization and filament stability; the mRNA content of Arpc3 and Lcp1 increased by 3- and 5-fold, respectively, but the levels of Tmod1 and Svil decreased by 2- and 5-fold, respectively. However, gravitational disuse did not result in changes in the mRNA content of Arpc3, Tmod1, Svil or Lcp1. Anti-orthostatic suspension for 6 hours resulted in a decrease in the mRNA content of alpha-actinin-4 (Actn4) and alpha-actinin-1 (Actn1) in group "HS" compared with that of group "C" by 25% and 30%, respectively, as well as a decrease and increase in the ACTN4 protein content in the membrane and cytoplasmic fractions, respectively. Lecithin injection resulted in an increase in the Actn1 and Actn4 mRNA content in group "C+L" by 1.5-fold and more than 2-fold, respectively, compared with the levels in group "C". Moreover, in group "HS+L", the mRNA content did not change in these genes compared with the levels in group "C+L", and the ACTN4 protein content in the membrane and cytoplasmic fractions also remained unchanged. Thus, lecithin prevented the reduction of Actn1 and Actn4 mRNA and the migration of ACTN4 from the cortical cytoskeleton to the cytoplasm.

Staphylococcus aureus nasal carriage in Ukraine: antibacterial resistance and virulence factor encoding genes.

PubMed

Netsvyetayeva, Irina; Fraczek, Mariusz; Piskorska, Katarzyna; Golas, Marlena; Sikora, Magdalena; Mlynarczyk, Andrzej; Swoboda-Kopec, Ewa; Marusza, Wojciech; Palmieri, Beniamino; Iannitti, Tommaso

2014-03-05

The number of studies regarding the incidence of multidrug resistant strains and distribution of genes encoding virulence factors, which have colonized the post-Soviet states, is considerably limited. The aim of the study was (1) to assess the Staphylococcus (S.) aureus nasal carriage rate, including Methicillin Resistant S. aureus (MRSA) strains in adult Ukrainian population, (2) to determine antibiotic resistant pattern and (3) the occurrence of Panton Valentine Leukocidine (PVL)-, Fibronectin-Binding Protein A (FnBPA)- and Exfoliative Toxin (ET)-encoding genes. Nasal samples for S. aureus culture were obtained from 245 adults. The susceptibility pattern for several classes of antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The virulence factor encoding genes, mecA, lukS-lukF, eta, etb, etd, fnbA, were detected by Polymerase Chain Reaction (PCR). The S. aureus nasal carriage rate was 40%. The prevalence of nasal MRSA carriage in adults was 3.7%. LukS-lukF genes were detected in over 58% of the strains. ET-encoding genes were detected in over 39% of the strains and the most prevalent was etd. The fnbA gene was detected in over 59% of the strains. All MRSA isolates tested were positive for the mecA gene. LukS-lukF genes and the etd gene were commonly co-present in MRSA, while lukS-lukF genes and the fnbA gene were commonly co-present in Methicillin Sensitive S. aureus (MSSA) isolates. No significant difference was detected between the occurrence of lukS-lukF genes (P > 0.05) and the etd gene (P > 0.05) when comparing MRSA and MSSA. The occurrence of the fnbA gene was significantly more frequent in MSSA strains (P < 0.05). In Ukraine, S. aureus is a common cause of infection. The prevalence of S. aureus nasal carriage in our cohort of patients from Ukraine was 40.4%. We found that 9.1% of the strains were classified as MRSA and all MRSA isolates tested positive for the mecA gene. We also observed a high prevalence of PVL- and ET- encoding genes among S. aureus nasal carriage strains. A systematic surveillance system can help prevent transmission and spread of drug resistant toxin producing S. aureus strains.

The SAM domain of ANKS6 has different interacting partners and mutations can induce different cystic phenotypes.

PubMed

Bakey, Zeineb; Bihoreau, Marie-Thérèse; Piedagnel, Rémi; Delestré, Laure; Arnould, Catherine; de Villiers, Alexandre d'Hotman; Devuyst, Olivier; Hoffmann, Sigrid; Ronco, Pierre; Gauguier, Dominique; Lelongt, Brigitte

2015-08-01

The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome

PubMed Central

Kunieda, T; Fujiyuki, T; Kucharski, R; Foret, S; Ament, S A; Toth, A L; Ohashi, K; Takeuchi, H; Kamikouchi, A; Kage, E; Morioka, M; Beye, M; Kubo, T; Robinson, G E; Maleszka, R

2006-01-01

Carbohydrate-metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate-rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate-metabolizing enzymes and 28 genes encoding lipid-metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose-methanol-choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes. PMID:17069632

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

PubMed Central

Ito, Teruyo; Ma, Xiao Xue; Takeuchi, Fumihiko; Okuma, Keiko; Yuzawa, Harumi; Hiramatsu, Keiichi

2004-01-01

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome. PMID:15215121

Mapping of Mcs30, a new mammary carcinoma susceptibility quantitative trait locus (QTL30) on rat chromosome 12: identification of fry as a candidate Mcs gene.

PubMed

Ren, Xuefeng; Graham, Jessica C; Jing, Lichen; Mikheev, Andrei M; Gao, Yuan; Lew, Jenny Pan; Xie, Hong; Kim, Andrea S; Shang, Xiuling; Friedman, Cynthia; Vail, Graham; Fang, Ming Zhu; Bromberg, Yana; Zarbl, Helmut

2013-01-01

Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.

Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norton, Jeanette M.; Klotz, Martin G; Stein, Lisa Y

2008-01-01

The complete genome of the ammonia-oxidizing bacterium, Nitrosospira multiformis (ATCC 25196T), consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2827 putative proteins. Of these, 2026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and N. eutropha were the best match for 42% of the predicted genes in N. multiformis. The genome contains three nearly identical copies of amo and hao gene clusters as large repeats. Distinguishing features compared to N. europaea include: the presencemore » of gene clusters encoding urease and hydrogenase, a RuBisCO-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced AOB genomes. Gene clusters encoding proteins associated with outer membrane and cell envelope functions including transporters, porins, exopolysaccharide synthesis, capsule formation and protein sorting/export were abundant. Numerous sensory transduction and response regulator gene systems directed towards sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate and cyanophycin storage and utilization were identified providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.« less

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021

Molecular cloning and characterization of alpha - galactosidase gene from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Moheer, Reyad Qaed Al; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

2015-09-01

Psychrophilic enzymes are proteins produced by psychrophilic organisms which recently are the limelight for industrial applications. A gene encoding α-galactosidase from a psychrophilic yeast, Glaciozyma antarctica PI12 which belongs to glycoside hydrolase family 27, was isolated and analyzed using several bioinformatic tools. The cDNA of the gene with the size of 1,404-bp encodes a protein with 467 amino acid residues. Predicted molecular weight of protein was 48.59 kDa and hence we name the gene encoding α-galactosidase as GAL48. We found that the predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal α-galactosidase.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

PubMed

Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

2010-01-01

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

EPA Science Inventory

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

PubMed

Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

2005-01-01

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

PubMed

Nunoura, Takuro; Hirayama, Hisako; Takami, Hideto; Oida, Hanako; Nishi, Shinro; Shimamura, Shigeru; Suzuki, Yohey; Inagaki, Fumio; Takai, Ken; Nealson, Kenneth H; Horikoshi, Koki

2005-12-01

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

PubMed Central

Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

2014-01-01

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments. PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

Curcumin regulates gene expression of insulin like growth factor, B-cell CLL/lymphoma 2 and antioxidant enzymes in streptozotocin induced diabetic rats

PubMed Central

2013-01-01

Background The effects of curcumin on the activities and gene expression of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (G-ST), B-cell CLL/lymphoma 2 (Bcl-2) and insulin like growth factor-1 (IGF-1) in diabetic rats were studied. Methods Twenty four rats were assigned to three groups (8 rats for each). Rats of first group were non diabetic and rats of the second group were rendered diabetic by streptozotocin (STZ). Both groups received vehicle, corn oil only (5 ml/kg body weight) and served as negative and positive controls, respectively. Rats of the third group were rendered diabetic and received oral curcumin dissolved in corn oil at a dose of 15 mg/5 ml/kg body weight for 6 weeks. Results Diabetic rats showed significant increase of blood glucose, thiobarbituric acid reactive substances (TBARS) and activities of all antioxidant enzymes with significant reduction of reduced glutathione (GSH) compare to the control non diabetic group. Gene expression of Bcl2, SOD, CAT, GPX and GST was increased significantly in diabetic untreated rats compare to the control non diabetic group. The administration of curcumin to diabetic rats normalized significantly their blood sugar level and TBARS values and increased the activities of all antioxidant enzymes and GSH concentration. In addition, curcumin treated rats showed significant increase in gene expression of IGF-1, Bcl2, SOD and GST compare to non diabetic and diabetic untreated rats. Conclusion Curcumin was antidiabetic therapy, induced hypoglycemia by up-regulation of IGF-1 gene and ameliorate the diabetes induced oxidative stress via increasing the availability of GSH, increasing the activities and gene expression of antioxidant enzymes and Bcl2. Further studies are required to investigate the actual mechanism of action of curcumin regarding the up regulation of gene expression of examined parameters. PMID:24364912

The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dyer, K.D.; Handen, J.S.; Rosenberg, H.F.

The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of {beta}-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire {beta}-galactoside bindingmore » site encoded by exon III. We have isolated CLC {beta}-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the {beta}-galactoside ligand. The most likely interpretation of these results suggests the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes. 35 refs., 3 figs.« less

Recombination and mutation of class II histocompatibility genes in wild mice.

PubMed

Wakeland, E K; Darby, B R

1983-12-01

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.

Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform.

PubMed

Schulte, W; Töpfer, R; Stracke, R; Schell, J; Martini, N

1997-04-01

Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide. The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B. napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from B. napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.

Opposite Effects of Cortisol on Consolidation of Temporal Sequence Memory during Waking and Sleep

ERIC Educational Resources Information Center

Wilhelm, Ines; Wagner, Ullrich; Born, Jan

2011-01-01

Memory functions involve three stages: encoding, consolidation, and retrieval. Modulating effects of glucocorticoids (GCs) have been consistently observed for declarative memory with GCs enhancing encoding and impairing retrieval, but surprisingly, little is known on how GCs affect memory consolidation. Studies in rats suggest a beneficial effect…

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

PubMed

Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

2012-05-01

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

PubMed Central

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

2014-01-01

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

PubMed

Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

2015-10-01

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

PubMed Central

Akagi, H; Patton, D E; Miledi, R

1989-01-01

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

Monosodium glutamate versus diet induced obesity in pregnant rats and their offspring.

PubMed

Afifi, M M; Abbas, Amr M

2011-06-01

We aim at determining the role of monosodium glutamate (MSG) compared with high caloric chow(HCC) in development of obesity in pregnant rats and their offspring. Ninety pregnant rats were divided into 3 groups, control, MSG and HCC fed. We determined energy intake, body weight (BW), abdominal fat, fat to body weight ratio, serum glucose, insulin, leptin, lipid profile, ob and leptin receptor-b gene expressions in pregnant rats and ob and leptin receptor-b gene expressions, serum insulin,glucose, leptin, triacylglycerides (TAG), total lipids (TL) and BW in offspring. Although daily energy intake and BW of MSG treated rats were lower than those of HCC fed rats, their abdominal fat and fat body weight ratio were higher. MSG or HCC increased Ob gene expression, leptin, insulin,LDL, cholesterol, total lipids (TL), glucose and decreased leptin receptor-b gene expression. In offspring of MSG treated rats, BW, serum glucose, insulin, leptin, TAG, TL and Ob gene expression increased and leptin receptor-b gene expression decreased whereas in offspring of HCC fed rats, serum insulin, leptin, Ob and leptin receptor-b gene expression increased but serum glucose, TAG, TL or BW did not change. We conclude that in pregnant rats, MSG, in spite of mild hypophagia, caused severe increase in fat body weight ratio, via leptin resistance, whereas, HCC increased BW and fat body weight ratio, due to hyperphagia with consequent leptin resistance. Moreover, maternal obesity in pregnancy, caused by MSG, has greater impact on offspring metabolism and BW than that induced by HCC.

Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

PubMed

Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Narnaware, S D; Mehta, S C; Singh, P K; Singh, Raghvendar; Tuteja, F C; Patil, N V

2015-06-01

The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

A mutation in a new gene bglJ, activates the bgl operon in Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giel, M.; Desnoyer, M.; Lopilato, J.

1996-06-01

A new mutation , bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic {beta}-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl{sup +} phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to amore » new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homology to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon. 56 refs., 3 figs., 3 tabs.« less

Two Closely Related Genes of Arabidopsis Encode Plastidial Cytidinediphosphate Diacylglycerol Synthases Essential for Photoautotrophic Growth1[C

PubMed Central

Haselier, André; Akbari, Hana; Weth, Agnes; Baumgartner, Werner; Frentzen, Margrit

2010-01-01

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth. PMID:20442275

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

PubMed

Henry, Romain; Bruneau, Emmanuelle; Gardan, Rozenn; Bertin, Stéphane; Fleuchot, Betty; Decaris, Bernard; Leblond-Bourget, Nathalie

2011-10-07

Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate.

PubMed

Horne, Irene; Sutherland, Tara D; Harcourt, Rebecca L; Russell, Robyn J; Oakeshott, John G

2002-07-01

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Detection of β-lactamase encoding genes in feces, soil and water from a Brazilian pig farm.

PubMed

Furlan, João Pedro Rueda; Stehling, Eliana Guedes

2018-01-10

β-lactam antibiotics are widely used for the treatment of different types of infections worldwide and the resistance to these antibiotics has grown sharply, which is of great concern. Resistance to β-lactams in gram-negative bacteria is mainly due to the production of β-lactamases, which are classified according to their functional activities. The aim of this study was to verify the presence of β-lactamases encoding genes in feces, soil, and water from a Brazilian pig farm. Different β-lactamases encoding genes were found, including bla CTX-M-Gp1 , bla CTX-M-Gp9 , bla SHV , bla OXA-1-like , bla GES , and bla VEB . The bla SHV and bla CTX-M-Gp1 genes have been detected in all types of samples, indicating the spread of β-lactam resistant bacteria among farm pigs and the environment around them. These results indicate that β-lactamase encoding genes belonging to the cloxacillinase, ESBL, and carbapenemase and they have high potential to spread in different sources, due to the fact that genes are closely related to mobile genetic elements, especially plasmids.

Episodic-like memory in the rat.

PubMed

Babb, Stephanie J; Crystal, Jonathon D

2006-07-11

A fundamental question in comparative cognition is whether animals remember unique, personal past experiences. It has long been argued that memories for specific events (referred to as episodic memory) are unique to humans. Recently, considerable evidence has accumulated to show that food-storing birds possess critical behavioral elements of episodic memory, referred to as episodic-like memory in acknowledgment of the fact that behavioral criteria do not assess subjective experiences. Here we show that rats have a detailed representation of remembered events and meet behavioral criteria for episodic-like memory. We provided rats with access to locations baited with distinctive (e.g., grape and raspberry) or nondistinctive (regular chow) flavors. Locations with a distinctive flavor replenished after a long but not a short delay, and locations with the nondistinctive flavor never replenished. One distinctive flavor was devalued after encoding its location by prefeeding that flavor (satiation) or by pairing it with lithium chloride (acquired taste aversion), while the other distinctive flavor was not devalued. The rats selectively decreased revisits to the devalued distinctive flavor but not to the nondevalued distinctive flavor. The present studies demonstrate that rats selectively encode the content of episodic-like memories.

New animal models reveal that coenzyme Q2 (Coq2) and placenta-specific 8 (Plac8) are candidate genes for the onset of type 2 diabetes associated with obesity in rats.

PubMed

Sasaki, Daiki; Kotoh, Jun; Watadani, Risa; Matsumoto, Kozo

2015-12-01

Obesity is a major risk factor for the onset of type 2 diabetes; however, little is known about the gene(s) involved. Therefore, we developed new animal models of obesity to search for diabetogenic genes associated with obesity. We generated double congenic rat strains with a hyperglycaemic quantitative trait locus (QTL) derived from the Otsuka Long-Evans Tokushima Fatty rat and a fa/fa (Lepr-/-) locus derived from the Zucker Fatty rat; phenotypic analysis for plasma glucose and insulin levels and RNA and protein levels were determined using reverse transcription quantitative PCR and Western blotting analyses, respectively. The double congenic strain F344-fa-nidd2 (Lepr-/- and Nidd2/of) exhibited significantly higher glucose levels and significantly lower hypoglycaemic response to insulin than the obese control strain F344-fa (Lepr-/-). These phenotypes were clearly observed in the obese strains but not in the lean strains. These results indicate that the Nidd2/of locus harbours a diabetogenic gene associated with obesity. We measured the expression of 60 genes in the Nidd2/of QTL region between the strains and found that the mRNA expression levels of five genes were significantly different between the strains under the condition of obesity. However, three of the five genes were differentially expressed in both obese and lean rats, indicating that these genes are not specific for the condition of obesity. Conversely, the other two genes, coenzyme Q2 (Coq2) and placenta-specific 8 (Plac8), were differentially expressed only in the obese rats, suggesting that these two genes are candidates for the onset of type 2 diabetes associated with obesity in rats.

The molecular genetics of Usher syndrome.

PubMed

Ahmed, Z M; Riazuddin, S; Riazuddin, S; Wilcox, E R

2003-06-01

Association of sensorineural deafness and progressive retinitis pigmentosa with and without a vestibular abnormality is the hallmark of Usher syndrome and involves at least 12 loci among three different clinical subtypes. Genes identified for the more commonly inherited loci are USH2A (encoding usherin), MYO7A (encoding myosin VIIa), CDH23 (encoding cadherin 23), PCDH15 (encoding protocadherin 15), USH1C (encoding harmonin), USH3A (encoding clarin 1), and USH1G (encoding SANS). Transcripts from all these genes are found in many tissues/cell types other than the inner ear and retina, but all are uniquely critical for retinal and cochlear cell function. Many of these protein products have been demonstrated to have direct interactions with each other and perform an essential role in stereocilia homeostasis.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

PubMed Central

Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

1989-01-01

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

Microarray Analyses of Genes Differentially Expressed by Diet (Black Beans and Soy Flour) during Azoxymethane-Induced Colon Carcinogenesis in Rats.

PubMed

Rondini, Elizabeth A; Bennink, Maurice R

2012-01-01

We previously demonstrated that black bean (BB) and soy flour (SF)-based diets inhibit azoxymethane (AOM)-induced colon cancer. The objective of this study was to identify genes altered by carcinogen treatment in normal-appearing colonic mucosa and those attenuated by bean feeding. Ninety-five male F344 rats were fed control (AIN) diets upon arrival. At 4 and 5 weeks, rats were injected with AOM (15 mg/kg) or saline and one week later administered an AIN, BB-, or SF-based diet. Rats were sacrificed after 31 weeks, and microarrays were conducted on RNA isolated from the distal colonic mucosa. AOM treatment induced a number of genes involved in immunity, including several MHC II-associated antigens and innate defense genes (RatNP-3, Lyz2, Pla2g2a). BB- and SF-fed rats exhibited a higher expression of genes involved in energy metabolism and water and sodium absorption and lower expression of innate (RatNP-3, Pla2g2a, Tlr4, Dmbt1) and cell cycle-associated (Cdc2, Ccnb1, Top2a) genes. Genes involved in the extracellular matrix (Col1a1, Fn1) and innate immunity (RatNP-3, Pla2g2a) were induced by AOM in all diets, but to a lower extent in bean-fed animals. This profile suggests beans inhibit colon carcinogenesis by modulating cellular kinetics and reducing inflammation, potentially by preserving mucosal barrier function.

Effects of apples and specific apple components on the cecal environment of conventional rats: role of apple pectin.

PubMed

Licht, Tine R; Hansen, Max; Bergström, Anders; Poulsen, Morten; Krath, Britta N; Markowski, Jaroslaw; Dragsted, Lars O; Wilcks, Andrea

2010-01-20

Our study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota. Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum Bacteroidetes, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum Firmicutes, and specific species belonging to the Clostridium Cluster XIVa.Quantitative real-time PCR confirmed a lower amount of given Bacteroidetes species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of Clostridium coccoides (belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals. Our findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and beta-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): II. Fetal rat brain.

PubMed

Pimenta, A F; Reinoso, B S; Levitt, P

1996-11-11

The limbic system-associated membrane protein (LAMP) is a 64-68 kDa neuronal surface glycoprotein expressed in cortical and subcortical regions of the limbic system of the adult and developing rat central nervous system (CNS). LAMP is a member of the immunoglobulin superfamily of cell adhesion molecules with three Ig domains and is highly conserved between rat and human. In this study, the temporal and spatial pattern of lamp gene expression during fetal rat development was analyzed by using Northern blot analysis and in situ hybridization. In Northern blot analysis, two lamp mRNA transcripts, 1.6 kb and 8.0 kb, identical in size to those present in the adult rat nervous system, were detected in developing neural tissue. In situ hybridization analysis showed close correlation, though not identity, between the expression of lamp mRNAs and the distribution of LAMP in limbic regions of the developing rat CNS, indicative of a more complex regulation of gene expression than was previously thought to be the case. The expression of lamp mRNAs is first detected on about embryonic day (E) 13. The hybridization signal is not seen in the proliferative ventricular zone at any level of the neuraxis, indicating that lamp is expressed in postmitotic neurons. In the cerebral cortex, lamp mRNAs are expressed in limbic cortical regions, such as the perirhinal cortex, prefrontal cortex, and cingulate cortex. In the hippocampus, the hybridization signal is observed in Ammon's horn by E18. The neostriatum, amygdaloid complex, and most hypothalamic areas express lamp mRNAs from early stages (E13-E14) in a pattern consistent with the onset of neurogenesis. The emerging patterns of lamp expression at the outset are similar to those seen in adult hypothalamus and dorsal thalamus. Although the hybridization signal is observed in some nonlimbic areas, including midbrain and hindbrain structures, intense labeling is evident in more classic limbic regions. The high levels of expression of lamp in limbic regions, beginning in early developmental stages, combined with the results of previous functional in vitro and in vivo studies, support a role for LAMP as a recognition molecule involved in the formation of limbic connections.

Identification of chitinolytic bacteria isolated from shrimp pond sediment and characterization of their chitinase encoding gene

NASA Astrophysics Data System (ADS)

Triwijayani, A. U.; Puspita, I. D.; Murwantoko; Ustadi

2018-03-01

Chitinolytic bacteria are a group of bacteria owning enzymes that able to hydrolyze chitin. Previously, we isolated chitinolytic bacteria from shrimp pond sediment in Bantul, Yogyakarta, and obtained five isolates showing high chitinolytic index named as isolate PT1, PT2, PT5, PT6 and PB2. The aims of this study were to identify chitinolytic bacteria isolated from shrimp pond sediment and to characterize the chitinase encoding gene from each isolate. The molecular technique was performed by amplification of 16S rDNA, amplification of chitinase encoding gene and sequence analysis. Two chitinolytic bacteria of PT1 and PT2 were similar to Aeromonas bivalvium strain D15, PT5 to Pseudomonas stutzeri strain BD-2.2.1, PT6 to Serratia marcescens strain FZSF02 and PB2 to Streptomyces misionensis strain OsiRt-1. The comparison of chitinase encoding gene between three isolates with those in Gen Bank shows that PT1 had similar sequences with the chi1 gene in Aeromonas sp. 17m, PT2 with chi1 gene in A. caviae (CB101) and PT6 with chiB gene in S. Marcescens (BJL200).

Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli

PubMed Central

Easton, Donna M.; Totsika, Makrina; Allsopp, Luke P.; Phan, Minh-Duy; Idris, Adi; Wurpel, Daniël J.; Sherlock, Orla; Zhang, Bing; Venturini, Carola; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.; Schembri, Mark A.

2011-01-01

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence. PMID:21687429

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

PubMed Central

Cecconi, Massimiliano; Parodi, Maria I.; Formisano, Francesco; Spirito, Paolo; Autore, Camillo; Musumeci, Maria B.; Favale, Stefano; Forleo, Cinzia; Rapezzi, Claudio; Biagini, Elena; Davì, Sabrina; Canepa, Elisabetta; Pennese, Loredana; Castagnetta, Mauro; Degiorgio, Dario; Coviello, Domenico A.

2016-01-01

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq™ Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status. PMID:27600940

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

PubMed Central

Yang, F; Curran, S C; Li, L S; Avarbock, D; Graf, J D; Chua, M M; Lu, G; Salem, J; Rubin, H

1997-01-01

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon. PMID:9335290

Harnessing pain heterogeneity and RNA transcriptome to identify blood-based pain biomarkers: a novel correlational study design and bioinformatics approach in a graded chronic constriction injury model.

PubMed

Grace, Peter M; Hurley, Daniel; Barratt, Daniel T; Tsykin, Anna; Watkins, Linda R; Rolan, Paul E; Hutchinson, Mark R

2012-09-01

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Activated Notch signaling cascade is correlated with stem cell differentiation toward absorptive progenitors after massive small bowel resection in a rat.

PubMed

Sukhotnik, Igor; Coran, Arnold G; Pollak, Yulia; Kuhnreich, Eviatar; Berkowitz, Drora; Saxena, Amulya K

2017-09-01

Notch signaling is thought to act to drive cell versification in the lining of the small intestine. The purpose of the present study was to evaluate the role of the Notch signaling pathway in stem cell differentiation in the late stages of intestinal adaptation after massive small bowel resection in a rat. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups of eight rats each: Sham rats underwent bowel transection and reanastomosis, while SBS rats underwent 75% small bowel resection. Rats were euthanized on day 14 Illumina's Digital Gene Expression (DGE) analysis was used to determine Notch signaling gene expression profiling. Notch-related gene and protein expression was determined using real-time PCR, Western blot analysis, and immunohistochemistry. From seven investigated Notch-related (by DGE analysis) genes, six genes were upregulated in SBS vs. control animals with a relative change in gene expression level of 20% or more. A significant upregulation of Notch signaling-related genes in resected animals was accompanied by a significant increase in Notch-1 protein levels (Western blot analysis) and a significant increase in the number of Notch1 and Hes1 (target gene)-positive cells (immunohistochemistry) compared with sham animals. Evaluation of cell differentiation has shown a strong increase in total number of absorptive cells (unchanged secretory cells) compared with control rats. In conclusion, 2 wk after bowel resection in rats, stimulated Notch signaling directs the crypt cell population toward absorptive progenitors. NEW & NOTEWORTHY This study provides novel insight into the mechanisms of cell proliferation following massive small bowel resection. We show that 2 wk after bowel resection in rats, enhanced stem cell activity was associated with stimulated Notch signaling pathway. We demonstrate that activated Notch signaling cascade directs the crypt cell population toward absorptive progenitors. Copyright © 2017 the American Physiological Society.

Direct Introduction of Genes into Rats and Expression of the Genes

NASA Astrophysics Data System (ADS)

Benvenisty, Nissim; Reshef, Lea

1986-12-01

A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.

Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons

PubMed Central

Pinto, Ashwin; Gillard, Samantha; Moss, Fraser; Whyte, Kathryn; Brust, Paul; Williams, Mark; Stauderman, Ken; Harpold, Michael; Lang, Bethan; Newsom-Davis, John; Bleakman, David; Lodge, David; Boot, John

1998-01-01

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the α1A cell line but not in the α1B, α1C, α1D, and α1E cell lines, establishing the α1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs. PMID:9653186

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection.

PubMed

McCarthy, Alex J; Martin, Patricia; Cloup, Emilie; Stabler, Richard A; Oswald, Eric; Taylor, Peter W

2015-09-01

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection

PubMed Central

McCarthy, Alex J.; Martin, Patricia; Cloup, Emilie; Stabler, Richard A.

2015-01-01

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate. PMID:26150540

Hippocampal gene expression in a rat model of depression after electroacupuncture at the Baihui and Yintang acupoints

PubMed Central

Duan, Dongmei; Yang, Xiuyan; Ya, Tu; Chen, Liping

2014-01-01

Preliminary basic research and clinical findings have demonstrated that electroacupuncture therapy exhibits positive effects in ameliorating depression. However, most studies of the underlying mechanism are at the single gene level; there are few reports regarding the mechanism at the whole-genome level. Using a rat genomic gene-chip, we profiled hippocampal gene expression changes in rats after electroacupuncture therapy. Electroacupuncture therapy alleviated depression-related manifestations in the model rats. Using gene-chip analysis, we demonstrated that electroacupuncture at Baihui (DU20) and Yintang (EX-HN3) regulates the expression of 21 genes. Real-time PCR showed that the genes Vgf, Igf2, Tmp32, Loc500373, Hif1a, Folr1, Nmb, and Rtn were upregulated or downregulated in depression and that their expression tended to normalize after electroacupuncture therapy. These results indicate that electroacupuncture at Baihui and Yintang modulates depression by regulating the expression of particular genes. PMID:25206746

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala.

PubMed

De la Rosa, J M; Pérez, J A; Gutiérrez, F; González, J M; Ruiz, T; Rodríguez, L

2001-11-01

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the beta-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5' region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3'-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. Copyright 2001 John Wiley & Sons, Ltd.