Pre-silencing of genes involved in the electron transport chain (ETC) pathway is associated with responsiveness to abatacept in rheumatoid arthritis.

PubMed

Derambure, C; Dzangue-Tchoupou, G; Berard, C; Vergne, N; Hiron, M; D'Agostino, M A; Musette, P; Vittecoq, O; Lequerré, T

2017-05-25

In the current context of personalized medicine, one of the major challenges in the management of rheumatoid arthritis (RA) is to identify biomarkers that predict drug responsiveness. From the European APPRAISE trial, our main objective was to identify a gene expression profile associated with responsiveness to abatacept (ABA) + methotrexate (MTX) and to understand the involvement of this signature in the pathophysiology of RA. Whole human genome microarrays (4 × 44 K) were performed from a first subset of 36 patients with RA. Data validation by quantitative reverse-transcription (qRT)-PCR was performed from a second independent subset of 32 patients with RA. Gene Ontology and WikiPathways database allowed us to highlight the specific biological mechanisms involved in predicting response to ABA/MTX. From the first subset of 36 patients with RA, a combination including 87 transcripts allowed almost perfect separation between responders and non-responders to ABA/MTX. Next, the second subset of patients 32 with RA allowed validation by qRT-PCR of a minimal signature with only four genes. This latter signature categorized 81% of patients with RA with 75% sensitivity, 85% specificity and 85% negative predictive value. This combination showed a significant enrichment of genes involved in electron transport chain (ETC) pathways. Seven transcripts from ETC pathways (NDUFA6, NDUFA4, UQCRQ, ATP5J, COX7A2, COX7B, COX6A1) were significantly downregulated in responders versus non-responders to ABA/MTX. Moreover, dysregulation of these genes was independent of inflammation and was specific to ABA response. Pre-silencing of ETC genes is associated with future response to ABA/MTX and might be a crucial key to susceptibility to ABA response.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

PubMed

Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

2015-01-01

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies.

PubMed

Taylor, Robert W; Pyle, Angela; Griffin, Helen; Blakely, Emma L; Duff, Jennifer; He, Langping; Smertenko, Tania; Alston, Charlotte L; Neeve, Vivienne C; Best, Andrew; Yarham, John W; Kirschner, Janbernd; Schara, Ulrike; Talim, Beril; Topaloglu, Haluk; Baric, Ivo; Holinski-Feder, Elke; Abicht, Angela; Czermin, Birgit; Kleinle, Stephanie; Morris, Andrew A M; Vassallo, Grace; Gorman, Grainne S; Ramesh, Venkateswaran; Turnbull, Douglass M; Santibanez-Koref, Mauro; McFarland, Robert; Horvath, Rita; Chinnery, Patrick F

2014-07-02

Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. To determine the molecular basis of multiple respiratory chain complex deficiencies. We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome.

PubMed

Kanabus, Marta; Fassone, Elisa; Hughes, Sean David; Bilooei, Sara Farahi; Rutherford, Tricia; Donnell, Maura O'; Heales, Simon J R; Rahman, Shamima

2016-05-01

There is growing interest in the use of the ketogenic diet (KD) to treat inherited metabolic diseases including mitochondrial disorders. However, neither the mechanism whereby the diet may be working, nor if it could benefit all patients with mitochondrial disease, is known. This study focusses on decanoic acid (C10), a component of the medium chain triglyceride KD, and a ligand for the nuclear receptor PPAR-γ known to be involved in mitochondrial biogenesis. The effects of C10 were investigated in primary fibroblasts from a cohort of patients with Leigh syndrome (LS) caused by nuclear-encoded defects of respiratory chain complex I, using mitochondrial respiratory chain enzyme assays, gene expression microarray, qPCR and flow cytometry. Treatment with C10 increased citrate synthase activity, a marker of cellular mitochondrial content, in 50 % of fibroblasts obtained from individuals diagnosed with LS in a PPAR-γ-mediated manner. Gene expression analysis and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst downregulating genes involved in glucose metabolism (PDK3, PDK4). PCK2, involved in blocking glucose metabolism, was upregulated, as was CAT, encoding catalase. Moreover, treatment with C10 also decreased oxidative stress in complex I deficient (rotenone treated) cells. However, since not all cells from subjects with LS appeared to respond to C10, prior cellular testing in vitro could be employed as a means for selecting individuals for subsequent clinical studies involving C10 preparations.

Novel cytochrome P450 genes, CYP6EB1 and CYP6EC1, are over-expressed in acrinathrin-resistant Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Cifuentes, D; Chynoweth, R; Guillén, J; De la Rúa, P; Bielza, P

2012-06-01

Control of Frankliniella occidentalis (Pergande) is a serious problem for agriculture all over the world because of the limited range of insecticides that are available. Insecticide resistance in F. occidentalis has been reported for all major insecticide groups. Our previous studies showed that cytochrome P450-mediated detoxification is a major mechanism responsible for insecticide resistance in this pest. Degenerate polymerase chain reaction was used to identify P450 genes that might be involved in acrinathrin resistance, in a laboratory population of F. occidentalis. Associated sequences were classified as belonging to the CYP4 and CYP6 families. Real-time quantitative polymerase chain reaction analyses revealed that two genes, CYP6EB1 and CYP6EC1, were over-expressed in adults and L2 larvae of the resistant population, when compared with the susceptible population, suggesting their possible involvement in resistance to acrinathrin.

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae.

PubMed

Vandenbosch, Davy; De Canck, Evelien; Dhondt, Inne; Rigole, Petra; Nelis, Hans J; Coenye, Tom

2013-12-01

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Involvement of Human Estrogen Related Receptor Alpha 1 (hERR 1) in Breast Cancer and Hormonally Insensitive Disease

DTIC Science & Technology

2000-08-01

SV40 early-to-late switch involves titration of cellular transcriptional repressors, Genes Dev. 7: 2206-19, 1993. 6. Bonnelye, E., Vanacker , J. M ...transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene, Mol Cell Biol. 17: 5400-9, 1997. 8. Vanacker , J. M ., Bonnelye, E...related receptor-alpha), Mol Endocrinol. 13: 764-73, 1999. 9. Vanacker , J. M ., Pettersson, K., Gustafsson, J. A., and Laudet, V. Transcriptional

Impact of Branched-Chain Amino Acid Catabolism on Fatty Acid and Alkene Biosynthesis in Micrococcus luteus.

PubMed

Surger, Maximilian J; Angelov, Angel; Stier, Philipp; Übelacker, Maria; Liebl, Wolfgang

2018-01-01

Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus , whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain α-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus . We also show how this knowledge can be used to engineer the isomeric composition and the chain lengths of the olefins produced by this organism.

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

PubMed

Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

2012-05-01

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

[Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction].

PubMed

Wang, F; Zhang, Y Q; Ding, J; Yu, L X

2017-10-18

To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome. Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type IV collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type IV collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome. A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study. Their genotypes were unknown. Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification. Genomic DNA was isolated from peripheral blood leukocytes in all the participants. Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction. When a deletion removed exon 1 of COL4A5 gene was identified, the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes, a promoter shared by COL4A5 and COL4A6 genes, and three reference genes in a single reaction. Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions. Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification. Deletions were identified in 6 of the 20 patients, including two large deletions removing the 5' part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6, two large deletions removing more than 30 exons of COL4A5 gene, one large deletion removing at least 1 exon of COL4A5 gene, and one small deletion involving 13 bps. No duplication was found. Our results show that multiplex competitive fluorescence polymerase chain reaction is a good alternative to classical techniques for large deletion genotyping in X-linked Alport syndrome.

Partial deficiency of isoleucine impairs root development and alters transcript levels of the genes involved in branched-chain amino acid and glucosinolate metabolism in Arabidopsis

PubMed Central

Liu, Dong

2013-01-01

Isoleucine is one of the branched-chain amino acids (BCAAs) that are essential substrates for protein synthesis in all organisms. Although the metabolic pathway for isoleucine has been well characterized in higher plants, it is not known whether it plays a specific role in plant development. In this study, an Arabidopsis mutant, lib (low isoleucine biosynthesis), that has defects in both cell proliferation and cell expansion processes during root development, was characterized. The lib mutant carries a T-DNA insertion in the last exon of the OMR1 gene that encodes a threonine deaminase/dehydratase (TD). TD catalyses the deamination and dehydration of threonine, which is the first and also the committed step in the biosynthesis of isoleucine. This T-DNA insertion results in a partial deficiency of isoleucine in lib root tissues but it does not affect its total protein content. Application of exogenous isoleucine or introduction of a wild-type OMR1 gene into the lib mutant can completely rescue the mutant phenotypes. These results reveal an important role for isoleucine in plant development. In addition, microarray analysis indicated that the partial deficiency of isoleucine in the lib mutant triggers a decrease in transcript levels of the genes encoding the major enzymes involved in the BCAA degradation pathway; the analysis also indicated that many genes involved in the biosynthesis of methionine-derived glucosinolates are up-regulated. PMID:23230023

Association mapping of starch chain length distribution and amylose content in pea (Pisum sativum L.) using carbohydrate metabolism candidate genes.

PubMed

Carpenter, Margaret A; Shaw, Martin; Cooper, Rebecca D; Frew, Tonya J; Butler, Ruth C; Murray, Sarah R; Moya, Leire; Coyne, Clarice J; Timmerman-Vaughan, Gail M

2017-08-01

Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea. To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel's r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways. The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

PubMed

Pequignot, M O; Desguerre, I; Dey, R; Tartari, M; Zeviani, M; Agostino, A; Benelli, C; Fouque, F; Prip-Buus, C; Marchant, D; Abitbol, M; Marsac, C

2001-05-04

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

Molecular Mechanisms of Increased Heart Rate in Shenxianshengmai-treated Bradycardia Rabbits.

PubMed

Liu, Zhou-Ying; Huang, Jian; Liu, Na-Na; Zheng, Min; Zhao, Tao; Zhao, Bu-Chang; Wang, Yi-Min; Pu, Jie-Lin

2017-01-20

The molecular mechanisms of Shenxianshengmai (SXSM), a traditional Chinese medicine, on bradycardia have been incompletely understood. The study tried to investigate the gene expression profile and proteomics of bradycardia rabbits' hearts after SXSM treatment. Twenty-four adult rabbits were randomly assigned in four groups: sham, model, model plus SXSM treatment, and sham plus SXSM treatment groups. Heart rate was recorded in all rabbits. Then, total RNA of atria and proteins of ventricle were isolated and quantified, respectively. Gene expression profiling was conducted by gene expression chip, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the results of gene expression chip. We used isobaric tags for elative and absolute quantitation and Western blotting to identify altered proteins after SXSM treatment. There was a constant decrease in the mean heart rate (32%, from 238 ± 6 beats/min to 149 ± 12 beats/min) after six weeks in model compared with that in sham group. This effect was partially reversed by 4-week SXSM treatment. Complementary DNA microarray demonstrated that the increased acetylcholinesterase and reduced nicotinic receptor were take responsibility for the increased heart rate. In addition, proteins involved in calcium handling and signaling were affected by SXSM treatment. Real-time RT-PCR verified the results from gene chip. Results from proteomics demonstrated that SXSM enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle in ventricular myocardium to improve ATP generation. Long-term SXSM stimulates sympathetic transmission by increasing the expression of acetylcholinesterase and reduces the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain.

Molecular Mechanisms of Increased Heart Rate in Shenxianshengmai-treated Bradycardia Rabbits

PubMed Central

Liu, Zhou-Ying; Huang, Jian; Liu, Na-Na; Zheng, Min; Zhao, Tao; Zhao, Bu-Chang; Wang, Yi-Min; Pu, Jie-Lin

2017-01-01

Background: The molecular mechanisms of Shenxianshengmai (SXSM), a traditional Chinese medicine, on bradycardia have been incompletely understood. The study tried to investigate the gene expression profile and proteomics of bradycardia rabbits’ hearts after SXSM treatment. Methods: Twenty-four adult rabbits were randomly assigned in four groups: sham, model, model plus SXSM treatment, and sham plus SXSM treatment groups. Heart rate was recorded in all rabbits. Then, total RNA of atria and proteins of ventricle were isolated and quantified, respectively. Gene expression profiling was conducted by gene expression chip, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the results of gene expression chip. We used isobaric tags for elative and absolute quantitation and Western blotting to identify altered proteins after SXSM treatment. Results: There was a constant decrease in the mean heart rate (32%, from 238 ± 6 beats/min to 149 ± 12 beats/min) after six weeks in model compared with that in sham group. This effect was partially reversed by 4-week SXSM treatment. Complementary DNA microarray demonstrated that the increased acetylcholinesterase and reduced nicotinic receptor were take responsibility for the increased heart rate. In addition, proteins involved in calcium handling and signaling were affected by SXSM treatment. Real-time RT-PCR verified the results from gene chip. Results from proteomics demonstrated that SXSM enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle in ventricular myocardium to improve ATP generation. Conclusions: Long-term SXSM stimulates sympathetic transmission by increasing the expression of acetylcholinesterase and reduces the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain. PMID:28091410

Development-related expression patterns of protein-coding and miRNA genes involved in porcine muscle growth.

PubMed

Wang, F J; Jin, L; Guo, Y Q; Liu, R; He, M N; Li, M Z; Li, X W

2014-11-27

Muscle growth and development is associated with remarkable changes in protein-coding and microRNA (miRNA) gene expression. To determine the expression patterns of genes and miRNAs related to muscle growth and development, we measured the expression levels of 25 protein-coding and 16 miRNA genes in skeletal and cardiac muscles throughout 5 developmental stages by quantitative reverse transcription-polymerase chain reaction. The Short Time-Series Expression Miner (STEM) software clustering results showed that growth-related genes were downregulated at all developmental stages in both the psoas major and longissimus dorsi muscles, indicating their involvement in early developmental stages. Furthermore, genes related to muscle atrophy, such as forkhead box 1 and muscle ring finger, showed unregulated expression with increasing age, suggesting a decrease in protein synthesis during the later stages of skeletal muscle development. We found that development of the cardiac muscle was a complex process in which growth-related genes were highly expressed during embryonic development, but they did not show uniform postnatal expression patterns. Moreover, the expression level of miR-499, which enhances the expression of the β-myosin heavy chain, was significantly different in the psoas major and longissimus dorsi muscles, suggesting the involvement of miR-499 in the determination of skeletal muscle fiber types. We also performed correlation analyses of messenger RNA and miRNA expression. We found negative relationships between miR-486 and forkhead box 1, and miR-133a and serum response factor at all developmental stages, suggesting that forkhead box 1 and serum response factor are potential targets of miR-486 and miR-133a, respectively.

[Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies].

PubMed

Scheps, Karen G; Varela, Viviana

Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2). In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

Heparan sulfate proteoglycans undergo differential expression alterations in right sided colorectal cancer, depending on their metastatic character.

PubMed

Fernández-Vega, Iván; García-Suárez, Olivia; García, Beatriz; Crespo, Ainara; Astudillo, Aurora; Quirós, Luis M

2015-10-20

Heparan sulfate proteoglycans (HSPGs) are complex molecules involved in the growth, invasion and metastatic properties of cancerous cells. This study analyses the alterations in the expression patterns of these molecules in right sided colorectal cancer (CRC), both metastatic and non-metastatic. Twenty right sided CRCs were studied. A transcriptomic approach was used, employing qPCR to analyze both the expression of the enzymes involved in heparan sulfate (HS) chains biosynthesis, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate (CS) chains, we include the study of the genes involved in the biosynthesis of these glycosaminoglycans. Immunohistochemical techniques were also used to analyze tissue expression of particular genes showing significant expression differences, of potential interest. Changes in proteoglycan core proteins differ depending on their location; those located intracellularly or in the extracellular matrix show very similar alteration patterns, while those located on the cell surface vary greatly depending on the nature of the tumor: glypicans 1, 3, 6 and betaglycan are affected in the non-metastatic tumors, whereas in the metastatic, only glypican-1 and syndecan-1 are modified, the latter showing opposing alterations in levels of RNA and of protein, suggesting post-transcriptional regulation in these tumors. Furthermore, in non-metastatic tumors, polymerization of glycosaminoglycan chains is modified, particularly affecting the synthesis of the tetrasaccharide linker and the initiation and elongation of CS chains, HS chains being less affected. Regarding the enzymes responsible for the modificaton of the HS chains, alterations were only found in non-metastatic tumors, affecting N-sulfation and the isoforms HS6ST1, HS3ST3B and HS3ST5. In contrast, synthesis of the CS chains suggests changes in epimerization and sulfation of the C4 and C2 in both types of tumor. Right sided CRCs show alterations in the expression of HSPGs, including the expression of the cell surface core proteins, many glycosiltransferases and some enzymes that modify the HS chains depending on the metastatic nature of the tumor, resulting more affected in non-metastatic ones. However, matrix proteoglycans and enzymes involved in CS fine structure synthesis are extensively modified independetly of the presence of lymph node metastasis.

Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

PubMed

Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

2016-01-01

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding β-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase1[OA

PubMed Central

Meesapyodsuk, Dauenpen; Balsevich, John; Reed, Darwin W.; Covello, Patrick S.

2007-01-01

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria. PMID:17172290

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

PubMed Central

Crosa, Jorge H.; Walsh, Christopher T.

2002-01-01

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium. PMID:12040125

Glomerular pathology in Alport syndrome: a molecular perspective

PubMed Central

Cosgrove, Dominic

2012-01-01

We have known for some time that mutations in the genes encoding 3 of the 6 type IV collagen chains are the underlying defect responsible for both X-linked (where the COL4A5 gene is involved) and autosomal (where either COL4A3 or COL4A4 genes are involved) Alport syndrome. The result of these mutations is the absence of the sub-epithelial network of all three chains in the glomerular basement membrane (GBM) resulting, at maturity, in a type IV collagen GBM network comprised of only α1(IV) and α2(IV) chains. The altered GBM functions adequately in early life. Eventually there is onset of proteinuria associated with the classic and progressive irregular thickening, thinning, and splitting of the GBM, which culminates in end stage renal failure. We have learned much about the molecular events associated with disease onset and progression through the study of animal models for Alport syndrome, and have identified some potential therapeutic approaches that may serve to delay the onset or slow the progression of the disease. This review focuses on where we are in our understanding of the disease, where we need to go to understand the molecular triggers that set the process in motion, and what emergent therapeutic approaches show promise for ameliorating disease progression in the clinic. PMID:21455721

Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

PubMed Central

Zeng, Mu-Heng; Liu, Sheng-Hong; Yang, Miao-Xian; Zhang, Ya-Jun; Liang, Jia-Yong; Wan, Xiao-Rong; Liang, Hong

2013-01-01

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. PMID:23880865

Posttransplantation lymphoproliferative disease involving the pituitary gland.

PubMed

Meriden, Zina; Bullock, Grant C; Bagg, Adam; Bonatti, Hugo; Cousar, John B; Lopes, M Beatriz; Robbins, Mark K; Cathro, Helen P

2010-11-01

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD. Copyright © 2010 Elsevier Inc. All rights reserved.

DETECTION OF PATHOGENS IN DRINKING WATER (SEER 2)

EPA Science Inventory

Project investigators developed a polymerase chain reaction (PCR)-based technique to detect E. coli 0157:H7 cells in environmental samples using previously reported PCR primers for the specific detection of genes involved in biosynthesis of 0157 polysacchari...

Continued Expression of Neonatal Myosin Heavy Chain in Adult Dystrophic Skeletal Muscle

NASA Astrophysics Data System (ADS)

Bandman, Everett

1985-02-01

The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.

Strategies for manipulation of oxygen utilization by the electron transfer chain in microbes for metabolic engineering purposes.

PubMed

Bennett, George N; San, Ka-Yiu

2017-05-01

Microaerobic growth is of importance in ecological niches, pathogenic infections and industrial production of chemicals. The use of low levels of oxygen enables the cell to gain energy and grow more robustly in the presence of a carbon source that can be oxidized and provide electrons to the respiratory chain in the membrane. A considerable amount of information is available on the genes and proteins involved in respiratory growth and the regulation of genes involved in aerobic and anaerobic metabolism. The dependence of regulation on sensing systems that respond to reduced quinones (e.g. ArcB) or oxygen levels that affect labile redox components of transcription regulators (Fnr) are key in understanding the regulation. Manipulation of the amount of respiration can be difficult to control in dense cultures or inadequately mixed reactors leading to inhomogeneous cultures that may have lower than optimal performance. Efforts to control respiration through genetic means have been reported and address mutations affecting components of the electron transport chain. In a recent report completion for intermediates of the ubiquinone biosynthetic pathway was used to dial the level of respiration vs lactate formation in an aerobically grown E. coli culture.

Gene expression analysis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia.

PubMed

Burgos, Carmen Mesas; Uggla, Andreas Ringman; Fagerström-Billai, Fredrik; Eklöf, Ann-Christine; Frenckner, Björn; Nord, Magnus

2010-07-01

Pulmonary hypoplasia and persistent pulmonary hypertension are the main causes of mortality and morbidity in newborns with congenital diaphragmatic hernia (CDH). Nitrofen is well known to induce CDH and lung hypoplasia in a rat model, but the mechanism remains unknown. To increase the understanding of the underlying pathogenesis of CDH, we performed a global gene expression analysis using microarray technology. Pregnant rats were given 100 mg nitrofen on gestational day 9.5 to create CDH. On day 21, fetuses after nitrofen administration and control fetuses were removed; and lungs were harvested. Global gene expression analysis was performed using Affymetrix Platform and the RAE 230 set arrays. For validation of microarray data, we performed real-time polymerase chain reaction and Western blot analysis. Significantly decreased genes after nitrofen administration included several growth factors and growth factors receptors involved in lung development, transcription factors, water and ion channels, and genes involved in angiogenesis and extracellular matrix. These results could be confirmed with real-time polymerase chain reaction and protein expression studies. The pathogenesis of lung hypoplasia and CDH in the nitrofen model includes alteration at a molecular level of several pathways involved in lung development. The complexity of the nitrofen mechanism of action reminds of human CDH; and the picture is consistent with lung hypoplasia and vascular disease, both important contributors to the high mortality and morbidity in CDH. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques to stimulate pre- and postnatal lung growth. Copyright 2010 Elsevier Inc. All rights reserved.

Genes and proteins involved in bacterial magnetic particle formation.

PubMed

Matsunaga, Tadashi; Okamura, Yoshiko

2003-11-01

Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

INS-gene mutations: from genetics and beta cell biology to clinical disease.

PubMed

Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

2015-04-01

A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. Copyright © 2014 Elsevier Ltd. All rights reserved.

INS-gene mutations: From genetics and beta cell biology to clinical disease

PubMed Central

Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

2015-01-01

A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. PMID:25542748

Sarcomere protein gene mutations and inherited heart disease: a beta-cardiac myosin heavy chain mutation causing endocardial fibroelastosis and heart failure.

PubMed

Kamisago, Mitsuhiro; Schmitt, Joachim P; McNamara, Dennis; Seidman, Christine; Seidman, J G

2006-01-01

Inherited human cardiomyopathies often lead to heart failure. A common feature of these conditions is that affected individuals can express the disease causing mutations for many years without showing clinical signs of the disease. Previous studies have demonstrated that sarcomere protein gene mutations can cause either dilated cardiomyopathy or hypertrophic cardiomyopathy. Here we demonstrate that the Arg442His missense mutation in beta-cardiac myosin heavy chain (betaMHC) causes dilated cardiomyopathy, endocardial fibroelastosis and heart failure at a very early age. Using standard genetic engineering tools we and others have made murine models by introducing human disease causing mutations into mice. The central hypothesis of these studies has been that by identifying the pathophysiological pathways activated by these mutations we can define enzymatic activities that are modified during the disease process and which may be involved in pathways that involve more common forms of cardiac disease. Murine models bearing different mutant myosins are being used to address whether each disease causing mutant betaMHC activates the same or different cellular pathways. Dissecting the molecular pathways modulated by mutations in sarcomere protein genes as well as other genes has already demonstrated that there are multiple pathways leading to cardiac remodelling and heart failure. Defining the mechanisms by which mutations in the same genes activate different cellular pathways remains an important question.

The Geonomic Organization of the CD28 Gene. Implications for the Regulation of CD28 mRNA Expression and Heterogeneity

DTIC Science & Technology

1990-07-01

doanrmsialecgtonptcelsvstsrtinrnsoa Partial primary structure of the alpha and beta chains of human tdomn ctmvity nat Nrento 320 ticl levsis.ti tasoa...L. Moretta. and C. MW. Croce. tlon and RNA splicing defects in five cloned j6- thalassaemia genes. 1987. Tp44 molecules Involved In antigen-independent T cell acti- Na t ure 302:59 1.

Mitochondrial myopathies.

PubMed

DiMauro, Salvatore

2006-11-01

Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus).

PubMed

Parra, Zuly E; Arnold, Tamara; Nowak, Melissa A; Hellman, Lars; Miller, Robert D

2006-01-01

TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

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

Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

2010-01-07

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3more » (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.« less

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

PubMed

Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

2010-02-01

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.

Activation of Two Different Resistance Mechanisms in Saccharomyces cerevisiae upon Exposure to Octanoic and Decanoic Acids▿ †

PubMed Central

Legras, J. L.; Erny, C.; Le Jeune, C.; Lollier, M.; Adolphe, Y.; Demuyter, C.; Delobel, P.; Blondin, B.; Karst, F.

2010-01-01

Medium-chain fatty acids (octanoic and decanoic acids) are well known as fermentation inhibitors. During must fermentation, the toxicity of these fatty acids is enhanced by ethanol and low pH, which favors their entrance in the cell, resulting in a decrease of internal pH. We present here the characterization of the mechanisms involved in the establishment of the resistance to these fatty acids. The analysis of the transcriptome response to the exposure to octanoic and decanoic acids revealed that two partially overlapping mechanisms are activated; both responses share many genes with an oxidative stress response, but some key genes were activated differentially. The transcriptome response to octanoic acid stress can be described mainly as a weak acid response, and it involves Pdr12p as the main transporter. The phenotypic analysis of knocked-out strains confirmed the role of the Pdr12p transporter under the control of WAR1 but also revealed the involvement of the Tpo1p major facilitator superfamily proteins (MFS) transporter in octanoic acid expulsion. In contrast, the resistance to decanoic acid is composite. It also involves the transporter Tpo1p and includes the activation of several genes of the beta-oxidation pathway and ethyl ester synthesis. Indeed, the induction of FAA1 and EEB1, coding for a long-chain fatty acyl coenzyme A synthetase and an alcohol acyltransferase, respectively, suggests a detoxification pathway through the production of decanoate ethyl ester. These results are confirmed by the sensitivity of strains bearing deletions for the transcription factors encoded by PDR1, STB5, OAF1, and PIP2 genes. PMID:20851956

A new missense mutation in the BCKDHB gene causes the classic form of maple syrup urine disease (MSUD).

PubMed

Miryounesi, Mohammad; Ghafouri-Fard, Soudeh; Goodarzi, Hamedreza; Fardaei, Majid

2015-05-01

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disease caused by mutations in the BCKDHA, BCKDHB, DBT and DLD genes, which encode the E1α, E1β, E2 and E3 subunits of the branched chain α ketoacid dehydrogenase (BCKD) complex, respectively. This complex is involved in the metabolism of branched-chain amino acids. In this study, we analyzed the DNA sequences of BCKDHA and BCKDHB genes in an infant who suffered from MSUD and died at the age of 6 months. We found a new missense mutation in exon 5 of BCKDHB gene (c.508C>T). The heterozygosity of the parents for the mentioned nucleotide change was confirmed by direct sequence analysis of the corresponding segment. Another missense mutation has been found in the same codon previously and shown by in silico analyses to be deleterious. This report provides further evidence that this amino acid change can cause classic MSUD.

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

PubMed

Li, Y; Spellerberg, M B; Stevenson, F K; Capra, J D; Potter, K N

1996-03-01

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

NASA Technical Reports Server (NTRS)

Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

1999-01-01

The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

Genes In Space-5

NASA Image and Video Library

2018-04-13

iss055e020319 (April 13, 2018) --- Flight Engineer Ricky Arnold processes of samples inside the Miniature Polymerase Chain Reaction (miniPCR) for the Genes In Space-5 experiment. The research gathered from Genes in Space-5 may be valuable in the development of procedures to maintain astronaut health and prevent an increased risk of cancer on deep space missions. The investigation also provides a deeper understanding of the human immune system, while giving student researchers a direct connection to the space program and offering hands-on educational experiences on Earth and promoting involvement in STEM fields.

DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

PubMed

Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

2015-01-01

Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

Comparative Genomics of Syntrophic Branched-Chain Fatty Acid Degrading Bacteria

PubMed Central

Narihiro, Takashi; Nobu, Masaru K.; Tamaki, Hideyuki; Kamagata, Yoichi; Sekiguchi, Yuji; Liu, Wen-Tso

2016-01-01

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading “Syntrophomonas wolfei subsp. methylbutyratica” strain JCM 14075T (=4J5T), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1T, and non-BCFA-metabolizing S. wolfei subsp. wolfei strain GöttingenT. We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism. PMID:27431485

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

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes.

PubMed

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke; Færgeman, Nils J; Kallipolitis, Birgitte H

The foodborne pathogen Listeria monocytogenes is the causative agent of the invasive disease listeriosis. Infection by L. monocytogenes involves bacterial crossing of the intestinal barrier and intracellular replication in a variety of host cells. The PrfA protein is the master regulator of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression in the intestinal environment. In the gastrointestinal phase of infection, the bacterium encounters a variety of antimicrobial agents, including medium- and long-chain free fatty acids that are commonly found in our diet and as active components of bile. Here we show that subinhibitory concentrations of specific antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Genetics of human sensitivity to ultraviolet radiation

NASA Astrophysics Data System (ADS)

Cleaver, James E.

1994-07-01

the major human health effects of solar and artificial UV light occur from the UVB and UVC wavelength ranges and involve a variety of short-term and long-term deleterious changes to the skin and eyes. the more important initial damage to cellular macromolecules involves dimerization of adjacent pyrimidines in DNA to produce cyclobutane pyrimidine dimes, (6-4) pyrimidine- pyrimidone, and (6-4) dewar photoproducts. these photoproducts can be repaired by a genetically regulated enzyme system (nucleotide excision repair) which removes oligonucleotides 29-30 nucleotides long that contain the photoproducts, and synthesizes replacement patches. At least a dozen gene products are involved in the process of recognizing photoproducts in DNA, altering local DNA helicity and cleaving the polynucleotide chain at defined positions either side of a photoproduct. Hereditary mutations in many of these genes are recognized in the human genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). Several of the gene products have other functions involving the regulation of gene transcription which accounts for the complex clinical presentation of repair deficient diseases that involve sensitivity of the skin and eyes to UV light, increased solar carcinogenesis (in XP), demyelination, and ganglial calcification (in CS), hair abnormalities (in TTD), and developmental and neurological abnormalities

Ecology, Diversity, and Evolution of Magnetotactic Bacteria

PubMed Central

Bazylinski, Dennis A.

2013-01-01

SUMMARY Magnetotactic bacteria (MTB) are widespread, motile, diverse prokaryotes that biomineralize a unique organelle called the magnetosome. Magnetosomes consist of a nano-sized crystal of a magnetic iron mineral that is enveloped by a lipid bilayer membrane. In cells of almost all MTB, magnetosomes are organized as a well-ordered chain. The magnetosome chain causes the cell to behave like a motile, miniature compass needle where the cell aligns and swims parallel to magnetic field lines. MTB are found in almost all types of aquatic environments, where they can account for an important part of the bacterial biomass. The genes responsible for magnetosome biomineralization are organized as clusters in the genomes of MTB, in some as a magnetosome genomic island. The functions of a number of magnetosome genes and their associated proteins in magnetosome synthesis and construction of the magnetosome chain have now been elucidated. The origin of magnetotaxis appears to be monophyletic; that is, it developed in a common ancestor to all MTB, although horizontal gene transfer of magnetosome genes also appears to play a role in their distribution. The purpose of this review, based on recent progress in this field, is focused on the diversity and the ecology of the MTB and also the evolution and transfer of the molecular determinants involved in magnetosome formation. PMID:24006473

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

PubMed Central

Fassah, Dilla Mareistia; Jeong, Jin Young

2018-01-01

Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition. PMID:29502393

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls.

PubMed

Fassah, Dilla Mareistia; Jeong, Jin Young; Baik, Myunggi

2018-04-01

This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition.

adhA in Aspergillus parasiticus Is Involved in Conversion of 5′-Hydroxyaverantin to Averufin

PubMed Central

Chang, Perng-Kuang; Yu, Jiujiang; Ehrlich, Kenneth C.; Boue, Stephen M.; Montalbano, Beverly G.; Bhatnagar, Deepak; Cleveland, Thomas E.

2000-01-01

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD+-NADP+, and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture. PMID:11055914

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

PubMed

Niederwanger, Michael; Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-08-11

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata , one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress

PubMed Central

Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-01-01

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails. PMID:28800079

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages.

PubMed

Shulse, Christine N; Allen, Eric E

2011-01-01

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart.

PubMed

Geng, Zhimin; Wang, Jue; Pan, Lulu; Li, Ming; Zhang, Jitai; Cai, Xueli; Chu, Maoping

2017-04-01

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

Gene encoding the human. beta. -hexosaminidase. beta. chain: Extensive homology of intron placement in the. alpha. - and. beta. -chain genes

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

Proia, R.L.

1988-03-01

Lysosomal {beta}-hexosaminidase is composed of two structurally similar chains, {alpha} and {beta}, that are the products of different genes. Mutations in either gene causing {beta}-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the {alpha} and {beta} chains, the {beta}-chain gene was isolated, and its organization was characterized. The {beta}-chain coding region is divided into 14 exons distributed over {approx}40 kilobases of DNA. Comparison with the {alpha}-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions.more » This extensive sharing of intron placement demonstrates that the {alpha} and {beta} chains evolved by way of the duplication of a common ancestor.« less

The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

PubMed Central

Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

2016-01-01

Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

Gene Therapy for Infectious Diseases

PubMed Central

Bunnell, Bruce A.; Morgan, Richard A.

1998-01-01

Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. Approaches to gene therapy for infectious diseases can be divided into three broad categories: (i) gene therapies based on nucleic acid moieties, including antisense DNA or RNA, RNA decoys, and catalytic RNA moieties (ribozymes); (ii) protein approaches such as transdominant negative proteins and single-chain antibodies; and (iii) immunotherapeutic approaches involving genetic vaccines or pathogen-specific lymphocytes. It is further possible that combinations of the aforementioned approaches will be used simultaneously to inhibit multiple stages of the life cycle of the infectious agent. PMID:9457428

The expanding phenotype of mitochondrial myopathy.

PubMed

DiMauro, Salvatore; Gurgel-Giannetti, Juliana

2005-10-01

Our understanding of mitochondrial diseases (defined restrictively as defects in the mitochondrial respiratory chain) continues to progress apace. In this review we provide an update of information regarding disorders that predominantly or exclusively affect skeletal muscle. Most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency, and mutations in genes that control mitochondrial DNA (mtDNA) abundance and structure such as POLG and TK2. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with altered lipid composition of the inner mitochondrial membrane, but a putative secondary impairment of the respiratory chain remains to be documented. Concerning the 'other genome', the role played by mutations in protein encoding genes of mtDNA in causing isolated myopathies has been confirmed. It has also been confirmed that mutations in tRNA genes of mtDNA can cause predominantly myopathic syndromes and - contrary to conventional wisdom - these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, myalgia, cramps, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

Irradiation at 660 nm modulates different genes central to wound healing in wounded and diabetic wounded cell models

NASA Astrophysics Data System (ADS)

Houreld, Nicolette N.

2014-02-01

Wound healing is a highly orchestrated process and involves a wide variety of cellular components, chemokines and growth factors. Laser irradiation has influenced gene expression and release of various growth factors, cytokines and extracellular matrix proteins involved in wound healing. This study aimed to determine the expression profile of genes involved in wound healing in wounded and diabetic wounded fibroblast cells in response to irradiation at a wavelength of 660 nm. Human skin fibroblast cells (WS1) were irradiated with a diode laser (wavelength 660 nm; fluence 5 J/cm2; power output 100 mW; power density 11 mW/cm2; spot size 9.1 cm2; exposure duration 7 min 35 s). Total RNA was isolated and 1 μg reverse transcribed into cDNA which was used as a template in real-time qualitative polymerase chain reaction (qPCR). Eighty four genes involved in wound healing (extracellular matrix and cell adhesion; inflammatory cytokines and chemokines; growth factors; and signal transduction) were evaluated in wounded and diabetic wounded cell models. Forty eight hours post-irradiation, 6 genes were significantly upregulated and 8 genes were down-regulated in irradiated wounded cells, whereas 1 gene was up-regulated and 33 genes down-regulated in irradiated diabetic wounded cells. Irradiation of stressed fibroblast cells to a wavelength of 660 nm and a fluence of 5 J/cm2 modulated the expression of different genes involved in wound healing in different cell models. Modulation of these genes leads to the effects of laser irradiation seen both in vivo and in vitro, and facilitates the wound healing process.

Natural selection of the major histocompatibility complex (Mhc) in Hawaiian honeycreepers (Drepanidinae)

USGS Publications Warehouse

Jarvi, S.I.; Tarr, C.L.; Mcintosh, C.E.; Atkinson, C.T.; Fleischer, R.C.

2004-01-01

The native Hawaiian honeycreepers represent a classic example of adaptive radiation and speciation, but currently face one the highest extinction rates in the world. Although multiple factors have likely influenced the fate of Hawaiian birds, the relatively recent introduction of avian malaria is thought to be a major factor limiting honeycreeper distribution and abundance. We have initiated genetic analyses of class II ?? chain Mhc genes in four species of honeycreepers using methods that eliminate the possibility of sequencing mosaic variants formed by cloning heteroduplexed polymerase chain reaction products. Phylogenetic analyses group the honeycreeper Mhc sequences into two distinct clusters. Variation within one cluster is high, with dN > d S and levels of diversity similar to other studies of Mhc (B system) genes in birds. The second cluster is nearly invariant and includes sequences from honeycreepers (Fringillidae), a sparrow (Emberizidae) and a blackbird (Emberizidae). This highly conserved cluster appears reminiscent of the independently segregating Rfp-Y system of genes defined in chickens. The notion that balancing selection operates at the Mhc in the honeycreepers is supported by transpecies polymorphism and strikingly high dN/dS ratios at codons putatively involved in peptide interaction. Mitochondrial DNA control region sequences were invariant in the i'iwi, but were highly variable in the 'amakihi. By contrast, levels of variability of class II ?? chain Mhc sequence codons that are hypothesized to be directly involved in peptide interactions appear comparable between i'iwi and 'amakihi. In the i'iwi, natural selection may have maintained variation within the Mhc, even in the face of what appears to a genetic bottleneck.

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

PubMed Central

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C.; Aviram, Micha; Parvari, Ruti

2011-01-01

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. PMID:21496787

Identification and characterization of six glycosyltransferases involved in the biosynthesis of a new bacterial exopolysaccharide in Paenibacillus elgii.

PubMed

Ou, Li; Ang, Li; Chujun, Zhang; Jingyu, Huang; Yongli, Meng; Shenjing, Yuan; Junhua, Huang; Xu, Gao; Yulong, Yao; Rui, Yin; Jinpan, Hu; Bin, Ding; Xiufang, Hu

2018-02-01

Paenibacillus elgii B69 produces a new xylose-containing exopolysaccharide (EPS) that effectively removes the pollutants from wastewater through flocculation. However, information about the biosynthesis of this EPS is limited. In this study, sequence analysis showed six putative glycosyltransferases (GTs) genes in polysaccharide gene clusters involved in glycosidic linkages of repeating units. Each gene was deleted and phenotypes were examined to understand the functions of these genes. Two of the genes were deleted successfully to encode a priming glucose GT and a side-chain xylose GT, but other genes were unsuccessfully deleted because of the accumulation of toxic intermediate products. The six genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified. The activity of GTs was analyzed through mass spectrometry by using the purified membrane fraction as a lipid carrier receptor after a hexasaccharide repeated unit was reconstructed in vitro. The specificities of six different GTs and the building order of the hexasaccharide were characterized. This study provided a basis for future research on the biosynthetic pathway of EPS in Paenibacillus or other genera.

Transcriptome profile analysis of floral sex determination in cucumber.

PubMed

Wu, Tao; Qin, Zhiwei; Zhou, Xiuyan; Feng, Zhuo; Du, Yalin

2010-07-15

Cucumber has been widely studied as a model for floral sex determination. In this investigation, we performed genome-wide transcriptional profiling of apical tissue of a gynoecious mutant (Csg-G) and the monoecious wild-type (Csg-M) of cucumber in an attempt to isolate genes involved in sex determination, using the Solexa technology. The profiling analysis revealed numerous changes in gene expression attributable to the mutation, which resulted in the down-regulation of 600 genes and the up-regulation of 143 genes. The Solexa data were confirmed by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR). Gene ontology (GO) analysis revealed that the differentially expressed genes were mainly involved in biogenesis, transport and organization of cellular component, macromolecular and cellular biosynthesis, localization, establishment of localization, translation and other processes. Furthermore, the expression of some of these genes depended upon the tissue and the developmental stage of the flowers of gynoecious mutant. The results of this study suggest two important concepts, which govern sex determination in cucumber. First, the differential expression of genes involved in plant hormone signaling pathways, such as ACS, Asr1, CsIAA2, CS-AUX1 and TLP, indicate that phytohormones and their crosstalk might play a critical role in the sex determination. Second, the regulation of some transcription factors, including EREBP-9, may also be involved in this developmental process. Copyright (c) 2010 Elsevier GmbH. All rights reserved.

Local recurrence as immunoglobulin G4 (IgG4)-related disease 10 years after radiotherapy to ocular adnexal extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue.

PubMed

Matsuo, Toshihiko; Ichimura, Kouichi; Yoshino, Tadashi

2011-01-01

In 2000, a 48-year-old woman developed a left orbital mass with lacrimal gland involvement and then, in 2003, a right orbital mass with lacrimal gland involvement, both of which were diagnosed as extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). She underwent 30 Gy external beam radiation to bilateral orbital lesions. The lymphoma cells in both lesions did not share the same clonality, as shown by amplification by polymerase chain reaction of the immunoglobulin heavy chain gene. Immunoglobulin light chain analysis by immunohistochemistry and messenger RNA in situ hybridization showed λ chain monotype in the left orbital lesion but κ chain monotype in the right orbital lesion. She developed recurrent left orbital mass with high uptake on fluorodeoxyglucose positron emission tomography fused with computed tomography in 2010, and excisional biopsy disclosed the formation of follicles and infiltration with immunoglobulin G4 (IgG4)-positive plasma cells mainly in interfollicular areas. The immunoglobulin light chain analysis showed the λ chain and κ chain bitype. With the immunohistopathological diagnosis of IgG4-related disease, the serum IgG4 level was found to show elevation at 376 mg/dL, and the patient chose observation. This is the first reported case of development of IgG4-related disease after bilataral orbital MALT lymphoma with external beam radiotherapy.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

PubMed Central

ter Schure, Eelko G.; Flikweert, Marcel T.; van Dijken, Johannes P.; Pronk, Jack T.; Verrips, C. Theo

1998-01-01

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production. PMID:9546164

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis.

PubMed

Chen, Sixue; Glawischnig, Erich; Jørgensen, Kirsten; Naur, Peter; Jørgensen, Bodil; Olsen, Carl-Erik; Hansen, Carsten H; Rasmussen, Hasse; Pickett, John A; Halkier, Barbara A

2003-03-01

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.

Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

DOE PAGES

Berg, Jörg; Mcdowell, Mindy; Jäck, Hans-Martin; ...

1990-01-01

Imore » mmunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ .t has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ -chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ -chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.« less

Protective pathways against colitis mediated by appendicitis and appendectomy

PubMed Central

Cheluvappa, R; Luo, A S; Palmer, C; Grimm, M C

2011-01-01

Appendicitis followed by appendectomy (AA) at a young age protects against inflammatory bowel disease (IBD). Using a novel murine appendicitis model, we showed that AA protected against subsequent experimental colitis. To delineate genes/pathways involved in this protection, AA was performed and samples harvested from the most distal colon. RNA was extracted from four individual colonic samples per group (AA group and double-laparotomy control group) and each sample microarray analysed followed by gene-set enrichment analysis (GSEA). The gene-expression study was validated by quantitative reverse transcription–polymerase chain reaction (RT–PCR) of 14 selected genes across the immunological spectrum. Distal colonic expression of 266 gene-sets was up-regulated significantly in AA group samples (false discovery rates < 1%; P-value < 0·001). Time–course RT–PCR experiments involving the 14 genes displayed down-regulation over 28 days. The IBD-associated genes tnfsf10, SLC22A5, C3, ccr5, irgm, ptger4 and ccl20 were modulated in AA mice 3 days after surgery. Many key immunological and cellular function-associated gene-sets involved in the protective effect of AA in experimental colitis were identified. The down-regulation of 14 selected genes over 28 days after surgery indicates activation, repression or de-repression of these genes leading to downstream AA-conferred anti-colitis protection. Further analysis of these genes, profiles and biological pathways may assist in developing better therapeutic strategies in the management of intractable IBD. PMID:21707591

Usage of DNA Fingerprinting Technology for Quality Control in Molecular Lab Bench Work.

PubMed

McIntosh, Linda Y; Lal, Janella E; Qin, Dahui

2018-01-01

One of the major quality assurance (QA) goals in many molecular laboratories is to avoid sample pipetting errors on the lab bench; especially when pipetting into multiwell plates. A pipetting error can cause a switch in patient samples, which can lead to recording the wrong results for the patient samples involved. Such pipetting errors are difficult to identify when it happens in lab bench work. DNA fingerprinting is a powerful tool in determining sample identities. Our laboratory has explored the usage of this technology in our QA process and successfully established that DNA fingerprinting can be used to monitor possible sample switch in gene rearrangement lab bench work. We use florescent light to quench the florescence in the gene rearrangement polymerase chain reaction products. After that, DNA fingerprinting technology is used to identify the sample DNA in the gene rearrangement polymerase chain reaction plate. The result is compared with the corresponding patient's blood sample DNA to determine whether there is a sample switch during the lab bench work.

Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

NASA Technical Reports Server (NTRS)

Baldwin, Kenneth M.; Haddad, Fadia

2002-01-01

The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome.

PubMed

Leigh, Margaret W; Pittman, Jessica E; Carson, Johnny L; Ferkol, Thomas W; Dell, Sharon D; Davis, Stephanie D; Knowles, Michael R; Zariwala, Maimoona A

2009-07-01

Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate(dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media)reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease),reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.

Transcriptional regulation by FOXP1, FOXP2, and FOXP4 dimerization.

PubMed

Sin, Cora; Li, Hongyan; Crawford, Dorota A

2015-02-01

FOXP1, FOXP2, and FOXP4 are three members of the FOXP gene subfamily of transcription factors involved in the development of the central nervous system. Previous studies have shown that the transcriptional activity of FOXP1/2/4 is regulated by homo- and heterodimerization. However, their transcriptional gene targets in the developing brain are still largely unknown. FOXP2 regulates the expression of many genes important in embryonic development, including WNT and Notch signaling pathways. In this study, we investigate whether dimerization of FOXP1/2/4 leads to differential expression of ten known FOXP2 target genes (CER1, SFRP4, WISP2, PRICKLE1, NCOR2, SNW1, NEUROD2, PAX3, EFNB3, and SLIT1). FOXP1/2/4 open-reading frames were stably transfected into HEK293 cells, and the expression level of these FOXP2 target genes was quantified using real-time polymerase chain reaction. Our results revealed that the specific combination of FOXP1/2/4 dimers regulates transcription of various FOXP2 target genes involved in early neuronal development.

Screening key candidate genes and pathways involved in insulinoma by microarray analysis.

PubMed

Zhou, Wuhua; Gong, Li; Li, Xuefeng; Wan, Yunyan; Wang, Xiangfei; Li, Huili; Jiang, Bin

2018-06-01

Insulinoma is a rare type tumor and its genetic features remain largely unknown. This study aimed to search for potential key genes and relevant enriched pathways of insulinoma.The gene expression data from GSE73338 were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified between insulinoma tissues and normal pancreas tissues, followed by pathway enrichment analysis, protein-protein interaction (PPI) network construction, and module analysis. The expressions of candidate key genes were validated by quantitative real-time polymerase chain reaction (RT-PCR) in insulinoma tissues.A total of 1632 DEGs were obtained, including 1117 upregulated genes and 514 downregulated genes. Pathway enrichment results showed that upregulated DEGs were significantly implicated in insulin secretion, and downregulated DEGs were mainly enriched in pancreatic secretion. PPI network analysis revealed 7 hub genes with degrees more than 10, including GCG (glucagon), GCGR (glucagon receptor), PLCB1 (phospholipase C, beta 1), CASR (calcium sensing receptor), F2R (coagulation factor II thrombin receptor), GRM1 (glutamate metabotropic receptor 1), and GRM5 (glutamate metabotropic receptor 5). DEGs involved in the significant modules were enriched in calcium signaling pathway, protein ubiquitination, and platelet degranulation. Quantitative RT-PCR data confirmed that the expression trends of these hub genes were similar to the results of bioinformatic analysis.The present study demonstrated that candidate DEGs and enriched pathways were the potential critical molecule events involved in the development of insulinoma, and these findings were useful for better understanding of insulinoma genesis.

[A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

PubMed

Kawahara, Hiroyuki; Yokosawa, Hideyoshi

2008-01-01

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

Brain region-specific gene expression changes after chronic intermittent ethanol exposure and early withdrawal in C57BL/6J mice

PubMed Central

Melendez, Roberto I.; McGinty, Jacqueline F.; Kalivas, Peter W.; Becker, Howard C.

2014-01-01

Neuroadaptations that participate in the ontogeny of alcohol dependence are likely a result of altered gene expression in various brain regions. The present study investigated brain region-specific changes in the pattern and magnitude of gene expression immediately following chronic intermittent ethanol (CIE) exposure and 8 hours following final ethanol exposure [i.e. early withdrawal (EWD)]. High-density oligonucleotide microarrays (Affymetrix 430A 2.0, Affymetrix, Santa Clara, CA, USA) and bioinformatics analysis were used to characterize gene expression and function in the prefrontal cortex (PFC), hippocampus (HPC) and nucleus accumbens (NAc) of C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA). Gene expression levels were determined using gene chip robust multi-array average followed by statistical analysis of microarrays and validated by quantitative real-time reverse transcription polymerase chain reaction and Western blot analysis. Results indicated that immediately following CIE exposure, changes in gene expression were strikingly greater in the PFC (284 genes) compared with the HPC (16 genes) and NAc (32 genes). Bioinformatics analysis revealed that most of the transcriptionally responsive genes in the PFC were involved in Ras/MAPK signaling, notch signaling or ubiquitination. In contrast, during EWD, changes in gene expression were greatest in the HPC (139 genes) compared with the PFC (four genes) and NAc (eight genes). The most transcriptionally responsive genes in the HPC were involved in mRNA processing or actin dynamics. Of the few genes detected in the NAc, the most representatives were involved in circadian rhythms. Overall, these findings indicate that brain region-specific and time-dependent neuroadaptive alterations in gene expression play an integral role in the development of alcohol dependence and withdrawal. PMID:21812870

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

PubMed

Legrand, Sylvain; Marque, Gilles; Blassiau, Christelle; Bluteau, Aurélie; Canoy, Anne-Sophie; Fontaine, Véronique; Jaminon, Odile; Bahrman, Nasser; Mautord, Julie; Morin, Julie; Petit, Aurélie; Baranger, Alain; Rivière, Nathalie; Wilmer, Jeroen; Delbreil, Bruno; Lejeune-Hénaut, Isabelle

2013-09-01

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively. Copyright © 2013 Elsevier GmbH. All rights reserved.

COL4A6 is dispensable for autosomal recessive Alport syndrome.

PubMed

Murata, Tomohiro; Katayama, Kan; Oohashi, Toshitaka; Jahnukainen, Timo; Yonezawa, Tomoko; Sado, Yoshikazu; Ishikawa, Eiji; Nomura, Shinsuke; Tryggvason, Karl; Ito, Masaaki

2016-07-05

Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role.

COL4A6 is dispensable for autosomal recessive Alport syndrome

PubMed Central

Murata, Tomohiro; Katayama, Kan; Oohashi, Toshitaka; Jahnukainen, Timo; Yonezawa, Tomoko; Sado, Yoshikazu; Ishikawa, Eiji; Nomura, Shinsuke; Tryggvason, Karl; Ito, Masaaki

2016-01-01

Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role. PMID:27377778

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

PubMed

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C; Aviram, Micha; Parvari, Ruti

2011-05-13

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

PubMed

Peters, Iain R; Helps, Chris R; Calvert, Emma L; Hall, Edward J; Day, Michael J

2005-01-01

To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

Biocide and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from the swine meat chain.

PubMed

Rizzotti, Lucia; Rossi, Franca; Torriani, Sandra

2016-12-01

In this study nine strains of Enterococcus faecalis and 12 strains of Enterococcus faecium, isolated from different sample types in the swine meat chain and previously characterized for the presence of antibiotic resistance genes, were examined for phenotypic tolerance to seven biocides (chlorexidine, benzalkonium chloride, triclosan, sodium hypochlorite, 2-propanol, formaldehyde and hydrogen peroxide) and resistance to nine antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol). Moreover, the presence of efflux system encoding genes qacA/B, qacC, qacE, qacEΔ1, emeA, and stress response genes, sigV and gsp65, involved in the tolerance to biocides, was analysed. Most strains were not tolerant to the biocides, but showed minimum inhibitory concentrations (MICs) higher than the recommended cut-off values for all the antibiotics tested, except for vancomycin and chloramphenicol. Only weak correlations, if any, were found between biocide and antibiotic resistance data. One E. faecalis strain was tolerant to triclosan and one E. faecium strain, with higher tolerance to chlorexidine than the other strains tested, was found to carry a qacA/B gene. Our results indicated that phenotypic resistance to antibiotics is very frequent in enterococcal isolates from the swine meat chain, but phenotypic tolerance to biocides is not common. On the other hand, the gene qacA/B was found for the first time in the species E. faecium, an indication of the necessity to adopt measures suitable to control the spread of biocide resistance determinants among enterococci. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

PubMed

Siddique, Bhuiyan Sharmin; Kinoshita, Shigeharu; Wongkarangkana, Chaninya; Asakawa, Shuichi; Watabe, Shugo

2016-06-01

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Light Regulation of the Arabidopsis Respiratory Chain. Multiple Discrete Photoreceptor Responses Contribute to Induction of Type II NAD(P)H Dehydrogenase Genes1

PubMed Central

Escobar, Matthew A.; Franklin, Keara A.; Svensson, Å. Staffan; Salter, Michael G.; Whitelam, Garry C.; Rasmusson, Allan G.

2004-01-01

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism. PMID:15333756

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation

PubMed Central

Hanada, Kazuharu; Ohsawa, Noboru

2017-01-01

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. PMID:28768733

The Molecular Basis of β-Thalassemia

PubMed Central

Thein, Swee Lay

2013-01-01

The β-thalassemias are characterized by a quantitative deficiency of β-globin chains underlaid by a striking heterogeneity of molecular defects. Although most of the molecular lesions involve the structural β gene directly, some down-regulate the gene through distal cis effects, and rare trans-acting mutations have also been identified. Most β-thalassemias are inherited in a Mendelian recessive fashion but there is a subgroup of β-thalassemia alleles that behave as dominant negatives. Unraveling the molecular basis of β-thalassemia has provided a paradigm for understanding of much of human genetics. PMID:23637309

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes.

PubMed

Macdonald, Lynn E; Karow, Margaret; Stevens, Sean; Auerbach, Wojtek; Poueymirou, William T; Yasenchak, Jason; Frendewey, David; Valenzuela, David M; Giallourakis, Cosmas C; Alt, Frederick W; Yancopoulos, George D; Murphy, Andrew J

2014-04-08

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.

Protective pathways against colitis mediated by appendicitis and appendectomy.

PubMed

Cheluvappa, R; Luo, A S; Palmer, C; Grimm, M C

2011-09-01

Appendicitis followed by appendectomy (AA) at a young age protects against inflammatory bowel disease (IBD). Using a novel murine appendicitis model, we showed that AA protected against subsequent experimental colitis. To delineate genes/pathways involved in this protection, AA was performed and samples harvested from the most distal colon. RNA was extracted from four individual colonic samples per group (AA group and double-laparotomy control group) and each sample microarray analysed followed by gene-set enrichment analysis (GSEA). The gene-expression study was validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) of 14 selected genes across the immunological spectrum. Distal colonic expression of 266 gene-sets was up-regulated significantly in AA group samples (false discovery rates < 1%; P-value < 0·001). Time-course RT-PCR experiments involving the 14 genes displayed down-regulation over 28 days. The IBD-associated genes tnfsf10, SLC22A5, C3, ccr5, irgm, ptger4 and ccl20 were modulated in AA mice 3 days after surgery. Many key immunological and cellular function-associated gene-sets involved in the protective effect of AA in experimental colitis were identified. The down-regulation of 14 selected genes over 28 days after surgery indicates activation, repression or de-repression of these genes leading to downstream AA-conferred anti-colitis protection. Further analysis of these genes, profiles and biological pathways may assist in developing better therapeutic strategies in the management of intractable IBD. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Differential expression of steroidogenic factors 1 and 2, cytochrome p450scc, and steroidogenic acute regulatory protein in human pancreas.

PubMed

Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Morimoto, Sumiko; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2008-08-01

The aim of this study was to investigate the expression of the 4 gene transcripts, steroidogenic factors 1 (SF-1) and 2 (SF-2), steroidogenic acute regulatory (StAR), and cytochrome P450 11A1, involved in the synthesis of steroid hormones in normal human pancreas. Total RNA was extracted from normal male (n = 5) and female (n = 5) samples, obtained from the organ donor program. The expression levels of SF-1, SF-2, StAR protein, and P450scc were assessed by reverse transcription-polymerase chain reaction and complemented with immunohistochemistry analysis. Polymerase chain reaction products amplification for all genes was present in both male and female samples, although differential expression was observed. The signals detected were much more evident in male than in female messenger RNA isolates for SF-1, SF-2, and StAR protein. The expression for P450scc was more intense in female samples. A similar pattern was observed in the immunohistochemical studies. Normal human pancreas expresses 4 gene transcripts involved in steroid synthesis similarly to steroidogenic organs. A distinctive characteristic is the sexually dimorphic expression of these factors. These data provide further evidence to support that the pancreas is a truly steroidogenic tissue, highlighting the presence of sex- and location-related differences in the expression of steroidogenic factors.

Homozygous deletion in MYL9 expands the molecular basis of megacystis-microcolon-intestinal hypoperistalsis syndrome.

PubMed

Moreno, Carolina Araujo; Sobreira, Nara; Pugh, Elizabeth; Zhang, Peng; Steel, Gary; Torres, Fábio Rossi; Cavalcanti, Denise Pontes

2018-05-01

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a severe disease characterized by functional obstruction in the urinary and gastrointestinal tract. The molecular basis of this condition started to be defined recently, and the genes related to the syndrome (ACTG2-heterozygous variant in sporadic cases; and MYH11 (myosin heavy chain 11), LMOD1 (leiomodin 1) and MYLK (myosin light chain (MLC) kinase)-autosomal recessive inheritance), encode proteins involved in the smooth muscle contraction, supporting a myopathic basis for the disease. In the present article, we described a family with two affected siblings with MMIHS born to consanguineous parents and the molecular investigation performed to define the genetic etiology. Previous whole exome sequencing of the affected child and parents did not identify a candidate gene for the disease in this family, but now we present a reanalysis of the data that led to the identification of a homozygous deletion encompassing the last exon of MYL9 (myosin regulatory light chain 9) in the affected individual. MYL9 gene encodes a regulatory myosin MLC and the phosphorylation of this protein is a crucial step in the contraction process of smooth muscle cell. Despite the absence of human or animal phenotype related to MYL9, a cause-effect relationship between MYL9 and the MMIHS seems biologically plausible. The present study reveals a strong candidate gene for autosomal recessive forms of MMIHS, expanding the molecular basis of this disease and reinforces the myopathic basis of this condition.

Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

PubMed

Eungwanichayapant, P D; Popluechai, S

2009-02-01

Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

A novel polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages.

PubMed

Cocolin, L; Manzano, M; Aggio, D; Cantoni, C; Comi, G

2001-05-01

A new molecular method consisting of polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) of a small fragment from the 16S rRNA gene identified the Micrococcaceae strains isolated from natural fermented Italian sausages. Lactic acid bacteria, total aerobic mesophilic flora, Enterobacteriaceae and faecal enterococci were also monitored. Micrococcaceaea control strains from international collections were used to optimise the method and 90 strains, isolated from fermented sausages, were identified by biochemical tests and PCR-DGGE. No differences were observed between the methods used. The results reported in this paper prove that Staphylococcus xylosus is the main bacterium involved in fermented sausage production, representing, from the tenth day of ripening, the only Micrococcaceaea species isolated.

Mutations of the Norrie gene in Korean ROP infants.

PubMed

Kim, Jeong Hun; Yu, Young Suk; Kim, Jiyeon; Park, Seong Sup

2002-12-01

The present study was conducted to evaluate if there is a Norrie disease gene (ND gene) mutation involved in the retinopathy of prematurity (ROP), and to identify the possibility of a genetic abnormality that may be linked to the presence of ROP. Nineteen premature Korean infants, with a low birth weight (1500 g or less) or low gestational age (32 weeks or less), were included in the study. Eighteen infants had ROP, and the other did not. Genomic DNA was isolated from the peripheral blood leukocytes of these patients, and all three exons and their flanking areas, all known ND gene mutations regions, were evaluated following amplification by a polymerase chain reaction, but no ND gene mutations were detected. Any disagreement between the relationship of ROP to the ND gene mutation will need to be clarified by further investigation.

Cancer Risk and Eicosanoid Production: Interaction between the Protective Effect of Long Chain Omega-3 Polyunsaturated Fatty Acid Intake and Genotype

PubMed Central

Lenihan-Geels, Georgia; Bishop, Karen S.; Ferguson, Lynnette R.

2016-01-01

Dietary inclusion of fish and fish supplements as a means to improve cancer prognosis and prevent tumour growth is largely controversial. Long chain omega-3 polyunsaturated fatty acids (LCn-3 PUFA), eicosapentaenoic acid and docosahexaenoic acid, may modulate the production of inflammatory eicosanoids, thereby influencing local inflammatory status, which is important in cancer development. Although in vitro studies have demonstrated inhibition of tumour cell growth and proliferation by LCn-3 PUFA, results from human studies have been mainly inconsistent. Genes involved in the desaturation of fatty acids, as well as the genes encoding enzymes responsible for eicosanoid production, are known to be implicated in tumour development. This review discusses the current evidence for an interaction between genetic polymorphisms and dietary LCn-3 PUFA in the risk for breast, prostate and colorectal cancers, in regards to inflammation and eicosanoid synthesis. PMID:26891335

McWRI1, a transcription factor of the AP2/SHEN family, regulates the biosynthesis of the cuticular waxes on the apple fruit surface under low temperature.

PubMed

Hao, Suxiao; Ma, Yiyi; Zhao, Shuang; Ji, Qianlong; Zhang, Kezhong; Yang, Mingfeng; Yao, Yuncong

2017-01-01

Cuticular waxes of plant and organ surfaces play an important role in protecting plants from biotic and abiotic stress and extending the freshness, storage time and shelf life in the post-harvest agricultural products. WRI1, a transcription factor of AP2/SHEN families, had been found to trigger the related genes taking part in the biosynthesis of seed oil in many plants. But whether WRI1 is involved in the biosynthesis of the cuticular waxes on the Malus fruits surface has been unclear. We investigated the changes of wax composition and structure, the related genes and WRI1 expression on Malus asiatica Nakai and sieversii fruits with the low temperature treatments, found that low temperature induced the up-regulated expression of McWRI1, which promoted gene expression of McKCS, McLACs and McWAX in very-long-chain fatty acid biosynthesis pathway, resulting in the accumulation of alkanes component and alteration of wax structure on the fruit surface. Corresponding results were verified in McWRI1 silenced by VIGS, and WRI1 silenced down-regulated the related genes on two kinds of fruits, it caused the diversity alteration in content of some alkanes, fatty acid and ester component in two kinds of fruits. We further conducted Y1H assay to find that McWRI1 transcription factor activated the promoter of McKCS, McLAC and McWAX to regulate their expression. These results demonstrated that McWRI1 is involved in regulating the genes related synthesis of very long chain fatty acid on surface of apple fruits in storage process, providing a highlight for improvement of the modified atmosphere storage of apple fruits.

McWRI1, a transcription factor of the AP2/SHEN family, regulates the biosynthesis of the cuticular waxes on the apple fruit surface under low temperature

PubMed Central

Ji, Qianlong; Zhang, Kezhong; Yang, Mingfeng

2017-01-01

Cuticular waxes of plant and organ surfaces play an important role in protecting plants from biotic and abiotic stress and extending the freshness, storage time and shelf life in the post-harvest agricultural products. WRI1, a transcription factor of AP2/SHEN families, had been found to trigger the related genes taking part in the biosynthesis of seed oil in many plants. But whether WRI1 is involved in the biosynthesis of the cuticular waxes on the Malus fruits surface has been unclear. We investigated the changes of wax composition and structure, the related genes and WRI1 expression on Malus asiatica Nakai and sieversii fruits with the low temperature treatments, found that low temperature induced the up-regulated expression of McWRI1, which promoted gene expression of McKCS, McLACs and McWAX in very-long-chain fatty acid biosynthesis pathway, resulting in the accumulation of alkanes component and alteration of wax structure on the fruit surface. Corresponding results were verified in McWRI1 silenced by VIGS, and WRI1 silenced down-regulated the related genes on two kinds of fruits, it caused the diversity alteration in content of some alkanes, fatty acid and ester component in two kinds of fruits. We further conducted Y1H assay to find that McWRI1 transcription factor activated the promoter of McKCS, McLAC and McWAX to regulate their expression. These results demonstrated that McWRI1 is involved in regulating the genes related synthesis of very long chain fatty acid on surface of apple fruits in storage process, providing a highlight for improvement of the modified atmosphere storage of apple fruits. PMID:29073205

Girdin Is an Intrinsic Regulator of Neuroblast Chain Migration in the Rostral Migratory Stream of the Postnatal Brain

PubMed Central

Wang, Yun; Kaneko, Naoko; Asai, Naoya; Enomoto, Atsushi; Isotani-Sakakibara, Mayu; Kato, Takuya; Asai, Masato; Murakumo, Yoshiki; Ota, Haruko; Hikita, Takao; Namba, Takashi; Kuroda, Keisuke; Kaibuchi, Kozo; Ming, Guo-li; Song, Hongjun; Sawamoto, Kazunobu; Takahashi, Masahide

2017-01-01

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed “chain migration.” The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin’s C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell–cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS. PMID:21632933

Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

PubMed Central

Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

2015-01-01

Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

PubMed

Weber, C; Hametner, C; Tuchscherer, A; Losand, B; Kanitz, E; Otten, W; Sauerwein, H; Bruckmaier, R M; Becker, F; Kanitz, W; Hammon, H M

2013-09-01

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A New Gene Expression Signature for Triple-Negative Breast Cancer Using Frozen Fresh Tissue before Neoadjuvant Chemotherapy

PubMed Central

Santuario-Facio, Sandra K; Cardona-Huerta, Servando; Perez-Paramo, Yadira X; Trevino, Victor; Hernandez-Cabrera, Francisco; Rojas-Martinez, Augusto; Uscanga-Perales, Grecia; Martinez-Rodriguez, Jorge L; Martinez-Jacobo, Lizeth; Padilla-Rivas, Gerardo; Muñoz-Maldonado, Gerardo; Gonzalez-Guerrero, Juan Francisco; Valero-Gomez, Javier; Vazquez-Guerrero, Ana L; Martinez-Rodriguez, Herminia G; Barboza-Quintana, Alvaro; Barboza-Quintana, Oralia; Garza-Guajardo, Raquel; Ortiz-Lopez, Rocio

2017-01-01

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer tumors. Comparisons between TNBC and non–triple-negative breast cancer (nTNBC) may help to differentiate key components involved in TNBC neoplasms. The purpose of the study was to analyze the expression profile of TNBC versus nTNBC tumors in a homogeneous population from northeastern Mexico. A prospective study of 50 patients (25 TNBC and 25 nTNBC) was conducted. Clinic parameters were equally distributed for TNBC and nTNBC: age at diagnosis (51 versus 47 years, p = 0.1), glucose level (107 mg/dl versus 104 mg/dl, p = 0.64), and body mass index (28 versus 29, p = 0.14). Core biopsies were collected for histopathological diagnosis and gene expression analysis. Total RNA was isolated and expression profiling was performed. Forty genes showed differential expression pattern in TNBC tumors. Among these, nine overexpressed genes (PRKX/PRKY, UGT8, HMGA1, LPIN1, HAPLN3, FAM171A1, BCL141A, FOXC1, and ANKRD11), and one underexpressed gene (ANX9) are involved in general metabolism. Based on this biochemical peculiarity and the overexpression of BCL11A and FOXC1 (involved in tumor growth and metastasis, respectively), we validated by quantitative polymerase chain reaction the expression profiles of seven genes out of the signature. In this report, a new gene signature for TNBC is proposed. To our knowledge, this is the first TNBC signature that describes genes involved in general metabolism. The findings may be pertinent for Mexican patients and require evaluation in other ethnic groups and populations. PMID:28474731

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Zinc finger protein rotund deficiency affects development of the thoracic leg in Bombyx mori.

PubMed

Zhou, Chun-Yan; Zha, Xing-Fu; Liu, Hua-Wei; Xia, Qing-You

2017-06-01

The insect limb develops from the imaginal disc or larval leg during metamorphosis. The molecular mechanisms involved in the development from the larval to the adult leg are poorly understood. Herein, we cloned the full length of a zinc finger gene rotund from Bombyx mori (Bmrn), which contained a 1419 bp open reading frame, and encoded a 473 amino acid protein. Reverse transcription polymerase chain reaction and Western blot analyses demonstrated that Bmrn was expressed at higher levels in the epidermis than in other tissues tested, and it showed a very high expression level during metamorphosis. Knock-down of Bmrn produced defects in the tarsus and pretarsus, including the fusion and reduction of tarsomeres, and the developmental arrest of pretarsus. Our data showed that Bmrn is involved in the formation of the tarsus and pretarsus, whereas its homologous gene in Drosophila has been shown to affect three tarsal segments (t2-t4), suggesting that the remodeling of the leg has involved changes in the patterning of gene regulation during evolution. © 2016 Institute of Zoology, Chinese Academy of Sciences.

MHC Class I Chain-Related Gene A Polymorphisms and Linkage Disequilibrium with HLA-B and HLA-C Alleles in Ocular Toxoplasmosis.

PubMed

Ayo, Christiane Maria; Camargo, Ana Vitória da Silveira; Frederico, Fábio Batista; Siqueira, Rubens Camargo; Previato, Mariana; Murata, Fernando Henrique Antunes; Silveira-Carvalho, Aparecida Perpétuo; Barbosa, Amanda Pires; Brandão de Mattos, Cinara de Cássia; de Mattos, Luiz Carlos

2015-01-01

This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05-4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22-0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

PubMed Central

MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

2008-01-01

C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

MHC Class I Chain-Related Gene A Polymorphisms and Linkage Disequilibrium with HLA-B and HLA-C Alleles in Ocular Toxoplasmosis

PubMed Central

Ayo, Christiane Maria; Camargo, Ana Vitória da Silveira; Frederico, Fábio Batista; Siqueira, Rubens Camargo; Previato, Mariana; Murata, Fernando Henrique Antunes; Silveira-Carvalho, Aparecida Perpétuo; Barbosa, Amanda Pires; Brandão de Mattos, Cinara de Cássia; de Mattos, Luiz Carlos

2015-01-01

This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05–4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22–0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population. PMID:26672749

A chain reaction approach to modelling gene pathways.

PubMed

Cheng, Gary C; Chen, Dung-Tsa; Chen, James J; Soong, Seng-Jaw; Lamartiniere, Coral; Barnes, Stephen

2012-08-01

BACKGROUND: Of great interest in cancer prevention is how nutrient components affect gene pathways associated with the physiological events of puberty. Nutrient-gene interactions may cause changes in breast or prostate cells and, therefore, may result in cancer risk later in life. Analysis of gene pathways can lead to insights about nutrient-gene interactions and the development of more effective prevention approaches to reduce cancer risk. To date, researchers have relied heavily upon experimental assays (such as microarray analysis, etc.) to identify genes and their associated pathways that are affected by nutrient and diets. However, the vast number of genes and combinations of gene pathways, coupled with the expense of the experimental analyses, has delayed the progress of gene-pathway research. The development of an analytical approach based on available test data could greatly benefit the evaluation of gene pathways, and thus advance the study of nutrient-gene interactions in cancer prevention. In the present study, we have proposed a chain reaction model to simulate gene pathways, in which the gene expression changes through the pathway are represented by the species undergoing a set of chemical reactions. We have also developed a numerical tool to solve for the species changes due to the chain reactions over time. Through this approach we can examine the impact of nutrient-containing diets on the gene pathway; moreover, transformation of genes over time with a nutrient treatment can be observed numerically, which is very difficult to achieve experimentally. We apply this approach to microarray analysis data from an experiment which involved the effects of three polyphenols (nutrient treatments), epigallo-catechin-3-O-gallate (EGCG), genistein, and resveratrol, in a study of nutrient-gene interaction in the estrogen synthesis pathway during puberty. RESULTS: In this preliminary study, the estrogen synthesis pathway was simulated by a chain reaction model. By applying it to microarray data, the chain reaction model computed a set of reaction rates to examine the effects of three polyphenols (EGCG, genistein, and resveratrol) on gene expression in this pathway during puberty. We first performed statistical analysis to test the time factor on the estrogen synthesis pathway. Global tests were used to evaluate an overall gene expression change during puberty for each experimental group. Then, a chain reaction model was employed to simulate the estrogen synthesis pathway. Specifically, the model computed the reaction rates in a set of ordinary differential equations to describe interactions between genes in the pathway (A reaction rate K of A to B represents gene A will induce gene B per unit at a rate of K; we give details in the "method" section). Since disparate changes of gene expression may cause numerical error problems in solving these differential equations, we used an implicit scheme to address this issue. We first applied the chain reaction model to obtain the reaction rates for the control group. A sensitivity study was conducted to evaluate how well the model fits to the control group data at Day 50. Results showed a small bias and mean square error. These observations indicated the model is robust to low random noises and has a good fit for the control group. Then the chain reaction model derived from the control group data was used to predict gene expression at Day 50 for the three polyphenol groups. If these nutrients affect the estrogen synthesis pathways during puberty, we expect discrepancy between observed and expected expressions. Results indicated some genes had large differences in the EGCG (e.g., Hsd3b and Sts) and the resveratrol (e.g., Hsd3b and Hrmt12) groups. CONCLUSIONS: In the present study, we have presented (I) experimental studies of the effect of nutrient diets on the gene expression changes in a selected estrogen synthesis pathway. This experiment is valuable because it allows us to examine how the nutrient-containing diets regulate gene expression in the estrogen synthesis pathway during puberty; (II) global tests to assess an overall association of this particular pathway with time factor by utilizing generalized linear models to analyze microarray data; and (III) a chain reaction model to simulate the pathway. This is a novel application because we are able to translate the gene pathway into the chemical reactions in which each reaction channel describes gene-gene relationship in the pathway. In the chain reaction model, the implicit scheme is employed to efficiently solve the differential equations. Data analysis results show the proposed model is capable of predicting gene expression changes and demonstrating the effect of nutrient-containing diets on gene expression changes in the pathway. One of the objectives of this study is to explore and develop a numerical approach for simulating the gene expression change so that it can be applied and calibrated when the data of more time slices are available, and thus can be used to interpolate the expression change at a desired time point without conducting expensive experiments for a large amount of time points. Hence, we are not claiming this is either essential or the most efficient way for simulating this problem, rather a mathematical/numerical approach that can model the expression change of a large set of genes of a complex pathway. In addition, we understand the limitation of this experiment and realize that it is still far from being a complete model of predicting nutrient-gene interactions. The reason is that in the present model, the reaction rates were estimated based on available data at two time points; hence, the gene expression change is dependent upon the reaction rates and a linear function of the gene expressions. More data sets containing gene expression at various time slices are needed in order to improve the present model so that a non-linear variation of gene expression changes at different time can be predicted.

Chemical event chain model of coupled genetic oscillators.

PubMed

Jörg, David J; Morelli, Luis G; Jülicher, Frank

2018-03-01

We introduce a stochastic model of coupled genetic oscillators in which chains of chemical events involved in gene regulation and expression are represented as sequences of Poisson processes. We characterize steady states by their frequency, their quality factor, and their synchrony by the oscillator cross correlation. The steady state is determined by coupling and exhibits stochastic transitions between different modes. The interplay of stochasticity and nonlinearity leads to isolated regions in parameter space in which the coupled system works best as a biological pacemaker. Key features of the stochastic oscillations can be captured by an effective model for phase oscillators that are coupled by signals with distributed delays.

Chemical event chain model of coupled genetic oscillators

NASA Astrophysics Data System (ADS)

Jörg, David J.; Morelli, Luis G.; Jülicher, Frank

2018-03-01

We introduce a stochastic model of coupled genetic oscillators in which chains of chemical events involved in gene regulation and expression are represented as sequences of Poisson processes. We characterize steady states by their frequency, their quality factor, and their synchrony by the oscillator cross correlation. The steady state is determined by coupling and exhibits stochastic transitions between different modes. The interplay of stochasticity and nonlinearity leads to isolated regions in parameter space in which the coupled system works best as a biological pacemaker. Key features of the stochastic oscillations can be captured by an effective model for phase oscillators that are coupled by signals with distributed delays.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

PubMed Central

1996-01-01

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

[Involvement of hydrogen peroxide in the regulation of coexpression of alternative oxidase and rotenone-insensitive NADH dehydrogenase in tomato leaves and calluses].

PubMed

Eprintsev, A T; Mal'tseva, E V; Shatskikh, A S; Popov, V N

2011-01-01

The involvement of active oxygen forms in the regulation of the expression of mitochondrial respiratory chain components, which are not related to energy storing, has been in vitro and in vivo studied in Lycopersicum esculentum L. The highest level of transcription of genes encoding alternative oxidase and NADH dehydrogenase has been observed in green tomato leaves. It has been shown that even low H2O2 concentrations activate both aoxlalpha and ndb1 genes, encoding alternative oxidase and external mitochondrial rotenone-insensitive NADH dehydrogenase, respectively. According to our results, in the case of an oxidative stress, alternative oxidase and NADH dehydrogenase are coexpressed in tomato plant tissues, and active oxygen forms serve as the secondary messengers of their coexpression.

Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide.

PubMed

Verdon, Julien; Labanowski, Jérome; Sahr, Tobias; Ferreira, Thierry; Lacombe, Christian; Buchrieser, Carmen; Berjeaud, Jean-Marc; Héchard, Yann

2011-04-01

Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires' disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid chain length in membrane were correlated with the increase in resistance to warnericin RK. Therefore, fatty acid profile seems to play a critical role in the sensitivity of L. pneumophila to warnericin RK. Copyright © 2010 Elsevier B.V. All rights reserved.

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

PubMed

Larsen, Nadja; Moslehi-Jenabian, Saloomeh; Werner, Birgit Brøsted; Jensen, Maiken Lund; Garrigues, Christel; Vogensen, Finn Kvist; Jespersen, Lene

2016-06-02

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production. Copyright © 2016 Elsevier B.V. All rights reserved.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection.

PubMed

Liu, Yan-Xia; Li, Fen-Xiang; Liu, Zhuan-Zhuan; Jia, Zhi-Rong; Zhou, Yan-He; Zhang, Hao; Yan, Hui; Zhou, Xian-Qiang; Chen, Xiao-Guang

2016-06-01

Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut. © 2016 Institute of Zoology, Chinese Academy of Sciences.

Integrative Analysis Reveals Regulatory Programs in Endometriosis

PubMed Central

Yang, Huan; Kang, Kai; Cheng, Chao; Mamillapalli, Ramanaiah; Taylor, Hugh S.

2015-01-01

Endometriosis is a common gynecological disease found in approximately 10% of reproductive-age women. Gene expression analysis has been performed to explore alterations in gene expression associated with endometriosis; however, the underlying transcription factors (TFs) governing such expression changes have not been investigated in a systematic way. In this study, we propose a method to integrate gene expression with TF binding data and protein–protein interactions to construct an integrated regulatory network (IRN) for endometriosis. The IRN has shown that the most regulated gene in endometriosis is RUNX1, which is targeted by 14 of 26 TFs also involved in endometriosis. Using 2 published cohorts, GSE7305 (Hover, n = 20) and GSE7307 (Roth, n = 36) from the Gene Expression Omnibus database, we identified a network of TFs, which bind to target genes that are differentially expressed in endometriosis. Enrichment analysis based on the hypergeometric distribution allowed us to predict the TFs involved in endometriosis (n = 40). This included known TFs such as androgen receptor (AR) and critical factors in the pathology of endometriosis, estrogen receptor α, and estrogen receptor β. We also identified several new ones from which we selected FOXA2 and TFAP2C, and their regulation was confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry (IHC). Further, our analysis revealed that the function of AR and p53 in endometriosis is regulated by posttranscriptional changes and not by differential gene expression. Our integrative analysis provides new insights into the regulatory programs involved in endometriosis. PMID:26134036

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

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.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

PubMed

Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

PubMed Central

Knowlton, Wendy M.; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury. PMID:28539870

Neutron Radiation Affects the Expression of Genes Involved in the Response to Auxin, Senescence and Oxidative Stress in Arabidopsis

NASA Astrophysics Data System (ADS)

Fortunati, A.; Tassone, P.; Migliaccio, F.

2008-06-01

Researches were conducted on the effect of neutron radiation on the expression of genes auxin activated or connected with the process of senescence in Arabidopsis plants. The research was done by applying the real-time polymerase chain reaction (PCR) technique. The results indicated that the auxin response factors (ARFs) genes are clearly downregulated, whereas the indolacetic acid-induced (Aux/IAAs) genes in some cases were upregulated. By contrast in the mutants for auxin transport aux1 and eir1 the ARFs genes were upregulated. In addition, both in the wildtype and mutants, some already known genes activated by stress and senescence were significantly upregulated. On the basis of these researches we conclude that the process of senescence induced by irradiation is, at least in part, controlled by the physiology of the hormone auxin.

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

A polymerase chain reaction-based methodology to detect gene doping.

PubMed

Carter, Adam; Flueck, Martin

2012-04-01

The non-therapeutic use of genes to enhance athletic performance (gene doping) is a novel threat to the world of sports. Skeletal muscle is a prime target of gene therapy and we asked whether we can develop a test system to produce and detect gene doping. Towards this end, we introduced a plasmid (pCMV-FAK, 3.8 kb, 50 μg) for constitutive expression of the chicken homologue for the regulator of muscle growth, focal adhesion kinase (FAK), via gene electro transfer in the anti-gravitational muscle, m. soleus, or gastrocnemius medialis of rats. Activation of hypertrophy signalling was monitored by assessing the ribosomal kinase p70S6K and muscle fibre cross section. Detectability of the introduced plasmid was monitored with polymerase chain reaction in deoxyribonucleic acids (DNA) from transfected muscle and serum. Muscle transfection with pCMV-FAK elevated FAK expression 7- and 73-fold, respectively, and increased mean cross section by 52 and 16% in targeted muscle fibres of soleus and gastrocnemius muscle 7 days after gene electro transfer. Concomitantly p70S6K content was increased in transfected soleus muscle (+110%). Detection of the exogenous plasmid sequence was possible in DNA and cDNA of muscle until 7 days after transfection, but not in serum except close to the site of plasmid deposition, 1 h after injection and surgery. The findings suggest that the reliable detection of gene doping in the immoral athlete is not possible unless a change in the current practice of tissue sampling is applied involving the collection of muscle biopsy close to the site of gene injection.

Gene expression analysis identifies new candidate genes associated with the development of black skin spots in Corriedale sheep.

PubMed

Peñagaricano, Francisco; Zorrilla, Pilar; Naya, Hugo; Robello, Carlos; Urioste, Jorge I

2012-02-01

The white coat colour of sheep is an important economic trait. For unknown reasons, some animals are born with, and others develop with time, black skin spots that can also produce pigmented fibres. The presence of pigmented fibres in the white wool significantly decreases the fibre quality. The aim of this work was to study gene expression in black spots (with and without pigmented fibres) and white skin by microarray techniques, in order to identify the possible genes involved in the development of this trait. Five unrelated Corriedale sheep were used and, for each animal, the three possible comparisons (three different hybridisations) between the three samples of interest were performed. Differential gene expression patterns were analysed using different t-test approaches. Most of the major genes with well-known roles in skin pigmentation, e.g. ASIP, MC1R and C-KIT, showed no significant difference in the gene expression between white skin and black spots. On the other hand, many of the differentially expressed genes (raw P-value < 0.005) detected in this study, e.g. C-FOS, KLF4 and UFC1, fulfil biological functions that are plausible to be involved in the formation of black spots. The gene expression of C-FOS and KLF4, transcription factors involved in the cellular response to external factors such as ultraviolet light, was validated by quantitative polymerase chain reaction (PCR). This exploratory study provides a list of candidate genes that could be associated with the development of black skin spots that should be studied in more detail. Characterisation of these genes will enable us to discern the molecular mechanisms involved in the development of this feature and, hence, increase our understanding of melanocyte biology and skin pigmentation. In sheep, understanding this phenomenon is a first step towards developing molecular tools to assist in the selection against the presence of pigmented fibres in white wool.

Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus.

PubMed

Shrihari, Rohinishree Yadahalli; Singh, Negi Pradeep

2012-02-01

Staphylococcus aureus survives well in different stress conditions. The ability of this organism to adapt to various stresses is the result of a complex regulatory response, which is attributed to regulation of multiple genes. The aims of the present study were (1) to develop a multiplex PCR for the detection of genes which are involved in stress adaptation (asp23, dnaK, and groEL); alternative sigma factor (sigB) and virulence determination (entB and spa) and (2) to study the expression of these genes during stress conditions for S. aureus culture collection strains (FRI 722 and ATCC 6538) and S. aureus food isolates at mRNA level using multiplex reverse transcription polymerase chain reaction (RT-PCR). During heat shock treatment groEL, dnaK, asp23, sodA, entB, spa, and sigB genes were up regulated up to 2.58, 2.07, 2.76, 2.55, 3.55, 2.71, and 2.62- folds, respectively, whereas in acid shock treatment, sodA and groEL were up regulated; dnaK was downregulated; and entB and sigB genes were not expressed in food isolates. Multiplex PCR assay standardized in this study offers an inexpensive alternative to uniplex PCR for detection of various virulence and stress response genes. This study is relevant to rapid and accurate detection of potential pathogenic S. aureus in foods. © 2012 Institute of Food Technologists®

JPRS Report, Science & Technology, USSR: Life Sciences.

DTIC Science & Technology

1988-02-12

polypeptide chain frag- ments inside protein membranes or on their surfaces using bacteriorhodopsin as the test object. Purple membranes, partially...outside the membrane or close to its surface . A model was developed from these data which involved folding of certain regions of bacteriorhodopsin...into hepatic endothelial and Kupffer cells. These findings point to the putative usefulness of the PLP approach in gene therapy. Figures h

Hydrocarbon activation under sulfate-reducing and methanogenic conditions proceeds by different mechanisms.

NASA Astrophysics Data System (ADS)

Head, Ian; Gray, Neil; Aitken, Caroline; Sherry, Angela; Jones, Martin; Larter, Stephen

2010-05-01

Microbial degradation of alkanes typically involves their conversion to fatty acids which are then catabolised by beta-oxidation. The critical step in this process is activation of the hydrocarbon. Under oxic conditions this is catalyzed by monooxygenase enzymes with the formation of long chain alcohols. In the absence of oxygen alternative alkane activation mechanisms have been observed or proposed. Fumarate addition to alkanes to form alkyl succinates is considered a central process in anaerobic hydrocarbon degradation. Comparative studies of crude oil degradation under sulphate-reducing and methanogenic conditions revealed distinctive patterns of compound class removal and metabolite formation. Alkyl succinates derived from C7 to C26 n-alkanes and branched chain alkanes were found in abundance in sulfate-reducing systems but these were not detected during methanogenic crude oil degradation. Only one other mechanism of alkane activation has been elucidated to date. This involves addition of carbon derived from bicarbonate/CO2 to C-3 of an alkane chain to form a 2-ethylalkane with subsequent removal of the ethyl group leading to the formation of a fatty acid 1 carbon shorter than the original alkane. 2-ethylalkanes have never been detected as metabolites of anaerobic alkane degradation and were not detected in crude oil-degrading methanogenic systems. Due to the range of alkanes present in crude oil it was not possible to infer the generation of C-odd acids from C-even alkanes which is characteristic of the C-3 carboxylation mechanism. Furthermore genes homologous to alkysuccinate synthetases were not detected in the methanogenic hydrocarbon degrading community by pyrosequencing of total DNA extracted from methanogenic enrichments cultures. beta-oxidation genes were detected and intriguingly, alcohol and aldehyde dehydrogenase genes were present. This offers the possibility that alkane activation in the methanogenic system does not proceed via acid metabolites, but may be initiated by an anaerobic hydroxylation reaction. This is not unprecedented and hydroxylation of ethylbenzene has been demonstrated. However the C-H bond dissociation energy of alkanes is typically considered too high to readily permit alkane hydroxylation. It is however clear that alkane activation in these methanogenic crude oil-degrading systems involves mechanisms other than the well-known fumarate-addition reactions.

Cutaneous lymphomatoid granulomatosis: correlation of clinical and biologic features.

PubMed

Beaty, M W; Toro, J; Sorbara, L; Stern, J B; Pittaluga, S; Raffeld, M; Wilson, W H; Jaffe, E S

2001-09-01

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus-associated B-cell lymphoproliferative disorder (EBV-BLPD), varying widely from an indolent process to an aggressive large cell lymphoma. The skin is the extrapulmonary organ most commonly involved in LYG. We studied 32 skin lesions from 20 patients with known pulmonary LYG, using immunohistochemistry, in situ hybridization for EBV, and polymerase chain reaction for the presence of antigen receptor gene rearrangements (IgH and TCR) to better define both the clinicopathologic spectrum and pathogenesis of the cutaneous lesions. We describe two distinct patterns of cutaneous involvement. Multiple erythematous dermal papules and/or subcutaneous nodules, with or without ulceration, were present in 17 patients (85%). These lesions demonstrate a marked angiocentric lymphohistiocytic infiltrate, composed predominantly of CD4-positive T-cells, with a high propensity for involving the subcutaneous tissues, and exhibiting angiodestruction, necrosis, and cytologic atypia. EBV-positive B-cells were detected in the nodules from five patients; clonal immunoglobulin heavy chain gene (IgH) rearrangements were detected by polymerase chain reaction in two patients. Multiple indurated, erythematous to white plaques were present in three patients (15%). The plaque lesions were negative for EBV and clonal IgH gene rearrangements in all cases studied. The clinical course of overall disease was variable, ranging from spontaneous regression without treatment (1 of 13; 7%), resolution with chemo/immunomodulatory therapy (8 of 13; 62%), and progression (4 of 13; 31%). The clinical and histopathologic features of cutaneous LYG are extremely diverse. However, the majority (85%) of the cutaneous lesions mirrors to some extent LYG in the lung, although EBV+ cells are less frequently identified. This subset of cases shows the histopathologic triad of angiodestruction with associated necrosis, panniculitis, and in some cases atypical lymphoid cells. The commonality of the histologic features in this group suggests a common pathophysiologic basis, possibly mediated by cytokines and chemokines induced by EBV. A small percentage of the lesions (15%) presented as indurated and atrophic plaques, and EBV was not identified in the small number of cases studied. The relationship of the plaque-like lesions to LYG remains uncertain. Whereas some cases of LYG regress spontaneously, most require therapy.

The Staphylococcus aureus Response to Unsaturated Long Chain Free Fatty Acids: Survival Mechanisms and Virulence Implications

PubMed Central

Kenny, John G.; Ward, Deborah; Josefsson, Elisabet; Jonsson, Ing-Marie; Hinds, Jason; Rees, Huw H.; Lindsay, Jodi A.; Tarkowski, Andrej; Horsburgh, Malcolm J.

2009-01-01

Staphylococcus aureus is an important human commensal and opportunistic pathogen responsible for a wide range of infections. Long chain unsaturated free fatty acids represent a barrier to colonisation and infection by S. aureus and act as an antimicrobial component of the innate immune system where they are found on epithelial surfaces and in abscesses. Despite many contradictory reports, the precise anti-staphylococcal mode of action of free fatty acids remains undetermined. In this study, transcriptional (microarrays and qRT-PCR) and translational (proteomics) analyses were applied to ascertain the response of S. aureus to a range of free fatty acids. An increase in expression of the σB and CtsR stress response regulons was observed. This included increased expression of genes associated with staphyloxanthin synthesis, which has been linked to membrane stabilisation. Similarly, up-regulation of genes involved in capsule formation was recorded as were significant changes in the expression of genes associated with peptidoglycan synthesis and regulation. Overall, alterations were recorded predominantly in pathways involved in cellular energetics. In addition, sensitivity to linoleic acid of a range of defined (sigB, arcA, sasF, sarA, agr, crtM) and transposon-derived mutants (vraE, SAR2632) was determined. Taken together, these data indicate a common mode of action for long chain unsaturated fatty acids that involves disruption of the cell membrane, leading to interference with energy production within the bacterial cell. Contrary to data reported for other strains, the clinically important EMRSA-16 strain MRSA252 used in this study showed an increase in expression of the important virulence regulator RNAIII following all of the treatment conditions tested. An adaptive response by S. aureus of reducing cell surface hydrophobicity was also observed. Two fatty acid sensitive mutants created during this study were also shown to diplay altered pathogenesis as assessed by a murine arthritis model. Differences in the prevalence and clinical importance of S. aureus strains might partly be explained by their responses to antimicrobial fatty acids. PMID:19183815

Transcriptome analysis of nitric oxide-responsive genes in upland cotton (Gossypium hirsutum).

PubMed

Huang, Juan; Wei, Hengling; Li, Libei; Yu, Shuxun

2018-01-01

Nitric oxide (NO) is an important signaling molecule with diverse physiological functions in plants. It is therefore important to characterize the downstream genes and signal transduction networks modulated by NO. Here, we identified 1,932 differentially expressed genes (DEGs) responding to NO in upland cotton using high throughput tag sequencing. The results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 25 DEGs showed good consistency. Gene Ontology (GO) and KEGG pathway were analyzed to gain a better understanding of these DEGs. We identified 157 DEGs belonging to 36 transcription factor (TF) families and 72 DEGs related to eight plant hormones, among which several TF families and hormones were involved in stress responses. Hydrogen peroxide and malondialdehyde (MDA) contents were increased, as well related genes after treatment with sodium nitroprusside (SNP) (an NO donor), suggesting a role for NO in the plant stress response. Finally, we compared of the current and previous data indicating a massive number of NO-responsive genes at the large-scale transcriptome level. This study evaluated the landscape of NO-responsive genes in cotton and identified the involvement of NO in the stress response. Some of the identified DEGs represent good candidates for further functional analysis in cotton.

Detecting long tandem duplications in genomic sequences.

PubMed

Audemard, Eric; Schiex, Thomas; Faraut, Thomas

2012-05-08

Detecting duplication segments within completely sequenced genomes provides valuable information to address genome evolution and in particular the important question of the emergence of novel functions. The usual approach to gene duplication detection, based on all-pairs protein gene comparisons, provides only a restricted view of duplication. In this paper, we introduce ReD Tandem, a software using a flow based chaining algorithm targeted at detecting tandem duplication arrays of moderate to longer length regions, with possibly locally weak similarities, directly at the DNA level. On the A. thaliana genome, using a reference set of tandem duplicated genes built using TAIR,(a) we show that ReD Tandem is able to predict a large fraction of recently duplicated genes (dS  <  1) and that it is also able to predict tandem duplications involving non coding elements such as pseudo-genes or RNA genes. ReD Tandem allows to identify large tandem duplications without any annotation, leading to agnostic identification of tandem duplications. This approach nicely complements the usual protein gene based which ignores duplications involving non coding regions. It is however inherently restricted to relatively recent duplications. By recovering otherwise ignored events, ReD Tandem gives a more comprehensive view of existing evolutionary processes and may also allow to improve existing annotations.

A mutagenesis study of a catalytic antibody

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

Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.

1991-01-01

The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and fourmore » Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.« less

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study

PubMed Central

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging. PMID:28626498

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study.

PubMed

Lehmann, Sylvia G; Bourgoin-Voillard, Sandrine; Seve, Michel; Rachidi, Walid

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging.

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.

PubMed

Shea, Patrick R; Virtaneva, Kimmo; Kupko, John J; Porcella, Stephen F; Barry, William T; Wright, Fred A; Kobayashi, Scott D; Carmody, Aaron; Ireland, Robin M; Sturdevant, Daniel E; Ricklefs, Stacy M; Babar, Imran; Johnson, Claire A; Graham, Morag R; Gardner, Donald J; Bailey, John R; Parnell, Michael J; Deleo, Frank R; Musser, James M

2010-03-09

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

J Genes for Heavy Chain Immunoglobulins of Mouse

NASA Astrophysics Data System (ADS)

Newell, Nanette; Richards, Julia E.; Tucker, Philip W.; Blattner, Frederick R.

1980-09-01

A 15.8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4Aλ phage vector system, was shown to contain the μ heavy chain constant region (CHμ ) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHμ gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila

PubMed Central

Williams, Thomas M.; Carroll, Sean B.

2009-01-01

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila. PMID:19652700

A novel regulatory defect in the branched-chain α-keto acid dehydrogenase complex due to a mutation in the PPM1K gene causes a mild variant phenotype of maple syrup urine disease.

PubMed

Oyarzabal, Alfonso; Martínez-Pardo, Mercedes; Merinero, Begoña; Navarrete, Rosa; Desviat, Lourdes R; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2013-02-01

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others. © 2012 WILEY PERIODICALS, INC.

Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

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

Kamb, A.; Weir, M.; Rudy, B.

1989-06-01

The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this methodmore » to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.« less

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.

PubMed

Shoji, Shisako; Hanada, Kazuharu; Ohsawa, Noboru; Shirouzu, Mikako

2017-09-07

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Molecular genetic analysis of podocyte genes in focal segmental glomerulosclerosis--a review.

PubMed

Löwik, M M; Groenen, P J; Levtchenko, E N; Monnens, L A; van den Heuvel, L P

2009-11-01

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

Genome-wide analysis of the genetic regulation of gene expression in human neutrophils

PubMed Central

Andiappan, Anand Kumar; Melchiotti, Rossella; Poh, Tuang Yeow; Nah, Michelle; Puan, Kia Joo; Vigano, Elena; Haase, Doreen; Yusof, Nurhashikin; San Luis, Boris; Lum, Josephine; Kumar, Dilip; Foo, Shihui; Zhuang, Li; Vasudev, Anusha; Irwanto, Astrid; Lee, Bernett; Nardin, Alessandra; Liu, Hong; Zhang, Furen; Connolly, John; Liu, Jianjun; Mortellaro, Alessandra; Wang, De Yun; Poidinger, Michael; Larbi, Anis; Zolezzi, Francesca; Rotzschke, Olaf

2015-01-01

Neutrophils are an abundant immune cell type involved in both antimicrobial defence and autoimmunity. The regulation of their gene expression, however, is still largely unknown. Here we report an eQTL study on isolated neutrophils from 114 healthy individuals of Chinese ethnicity, identifying 21,210 eQTLs on 832 unique genes. Unsupervised clustering analysis of these eQTLs confirms their role in inflammatory responses and immunological diseases but also indicates strong involvement in dermatological pathologies. One of the strongest eQTL identified (rs2058660) is also the tagSNP of a linkage block reported to affect leprosy and Crohn's disease in opposite directions. In a functional study, we can link the C allele with low expression of the β-chain of IL18-receptor (IL18RAP). In neutrophils, this results in a reduced responsiveness to IL-18, detected both on the RNA and protein level. Thus, the polymorphic regulation of human neutrophils can impact beneficial as well as pathological inflammatory responses. PMID:26259071

Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division.

PubMed

Bickert, Andreas; Kern, Paul; van Uelft, Martina; Herresthal, Stefanie; Ulas, Thomas; Gutbrod, Katharina; Breiden, Bernadette; Degen, Joachim; Sandhoff, Konrad; Schultze, Joachim L; Dörmann, Peter; Hartmann, Dieter; Bauer, Reinhard; Willecke, Klaus

2018-07-01

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of molecular markers as predictive factors of lymph node involvement in breast carcinoma.

PubMed

Paula, Luciana Marques; De Moraes, Luis Henrique Ferreira; Do Canto, Abaeté Leite; Dos Santos, Laurita; Martin, Airton Abrahão; Rogatto, Silvia Regina; De Azevedo Canevari, Renata

2017-01-01

Nodal status is the most significant independent prognostic factor in breast cancer. Identification of molecular markers would allow stratification of patients who require surgical assessment of lymph nodes from the large numbers of patients for whom this surgical procedure is unnecessary, thus leading to a more accurate prognosis. However, up to now, the reported studies are preliminary and controversial, and although hundreds of markers have been assessed, few of them have been used in clinical practice for treatment or prognosis in breast cancer. The purpose of the present study was to determine whether protein phosphatase Mg2+/Mn2+ dependent 1D, β-1,3-N-acetylglucosaminyltransferase, neural precursor cell expressed, developmentally down-regulated 9, prohibitin, phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5), phosphatidylinositol-5-phosphate 4-kinase type IIα, TRF1-interacting ankyrin-related ADP-ribose polymerase 2, BCL2 associated agonist of cell death, G2 and S-phase expressed 1 and PAX interacting protein 1 genes, described as prognostic markers in breast cancer in a previous microarray study, are also predictors of lymph node involvement in breast carcinoma Reverse transcription-quantitative polymerase chain reaction analysis was performed on primary breast tumor tissues from women with negative lymph node involvement (n=27) compared with primary tumor tissues from women with positive lymph node involvement (n=23), and was also performed on primary tumors and paired lymph node metastases (n=11). For all genes analyzed, only the PIK3R5 gene exhibited differential expression in samples of primary tumors with positive lymph node involvement compared with primary tumors with negative lymph node involvement (P=0.0347). These results demonstrate that the PIK3R5 gene may be considered predictive of lymph node involvement in breast carcinoma. Although the other genes evaluated in the present study have been previously characterized to be involved with the development of distant metastases, they did not have predictive potential.

Fusion gene profile of biphenotypic sinonasal sarcoma: an analysis of 44 cases.

PubMed

Fritchie, Karen J; Jin, Long; Wang, Xiaoke; Graham, Rondell P; Torbenson, Michael S; Lewis, Jean E; Rivera, Michael; Garcia, Joaquin J; Schembri-Wismayer, David J; Westendorf, Jennifer J; Chou, Margaret M; Dong, Jie; Oliveira, Andre M

2016-12-01

Biphenotypic sinonasal sarcoma (SNS) is a locally aggressive tumour that occurs in the sinonasal region. PAX3-MAML3 has recently been identified as a recurrent fusion gene event in this entity; however, a subset of tumours harbour alternative PAX3 rearrangement without the involvement of MAML3. In this study we sought to characterize the molecular profile of a large series of cases, with a special emphasis on tumours with alternative fusions. Forty-four examples of SNS were screened by fluorescence in-situ hybridization and reverse transcription polymerase chain reaction to better characterize its molecular profile and identify potential novel fusion genes. Twenty-four were positive for PAX3-MAML3 (55%), 15 showed rearrangements of PAX3 without MAML3 involvement (34%), one showed rearrangement of MAML3 without PAX3 involvement, and four were negative for the involvement of either gene (9%). Among 15 cases with PAX3 involvement only, three were found to harbour PAX3-FOXO1. Two of these cases arose in the nasal cavities of female patients (aged 31 and 47 years), and one showed bilateral involvement of the nasal cavities of a 35-year-old male. A fourth case involved the skull base of a 47-year-old male, and was positive for PAX3-NCOA1. Patients with fusion-negative tumours were slightly older. More than half of the SNSs in this series were positive for PAX3-MAML3. However, a subset of tumours may harbour alternative PAX3 fusion genes or show no involvement of PAX3. Except for a possible weak association between age and molecular profile, the overall morphological and immunophenotypic features of all cases seem to be similar. Because of the rarity of these tumours, the impact of the molecular profile on the clinical course of these tumours remains to be determined. © 2016 John Wiley & Sons Ltd.

Transcriptome response signatures associated with the overexpression of a mitochondrial uncoupling protein (AtUCP1) in tobacco.

PubMed

Laitz, Alessandra Vasconcellos Nunes; Acencio, Marcio Luis; Budzinski, Ilara G F; Labate, Mônica T V; Lemke, Ney; Ribolla, Paulo Eduardo Martins; Maia, Ivan G

2015-01-01

Mitochondrial inner membrane uncoupling proteins (UCP) dissipate the proton electrochemical gradient established by the respiratory chain, thus affecting the yield of ATP synthesis. UCP overexpression in plants has been correlated with oxidative stress tolerance, improved photosynthetic efficiency and increased mitochondrial biogenesis. This study reports the main transcriptomic responses associated with the overexpression of an UCP (AtUCP1) in tobacco seedlings. Compared to wild-type (WT), AtUCP1 transgenic seedlings showed unaltered ATP levels and higher accumulation of serine. By using RNA-sequencing, a total of 816 differentially expressed genes between the investigated overexpressor lines and the untransformed WT control were identified. Among them, 239 were up-regulated and 577 were down-regulated. As a general response to AtUCP1 overexpression, noticeable changes in the expression of genes involved in energy metabolism and redox homeostasis were detected. A substantial set of differentially expressed genes code for products targeted to the chloroplast and mainly involved in photosynthesis. The overall results demonstrate that the alterations in mitochondrial function provoked by AtUCP1 overexpression require important transcriptomic adjustments to maintain cell homeostasis. Moreover, the occurrence of an important cross-talk between chloroplast and mitochondria, which culminates in the transcriptional regulation of several genes involved in different pathways, was evidenced.

The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the PAX-5 gene.

PubMed

Iida, S; Rao, P H; Nallasivam, P; Hibshoosh, H; Butler, M; Louie, D C; Dyomin, V; Ohno, H; Chaganti, R S; Dalla-Favera, R

1996-12-01

The t(9;14)(p13;q32) translocation is associated with approximately 50% of lymphoplasmacytoid lymphoma (LPL), a subtype of B-cell non-Hodgkin's lymphoma (NHL). We cloned the chromosomal breakpoint of der (14) from an LPL case (1052) and showed that it involved a junction between 9p13 and the switch micro region of the Ig heavy chain locus (IgH) on 14q32. Using a YAC contig spanning 1.5 megabase (Mb), we determined that the 9p13 breakpoint in one case (1052) mapped within a 270-kb restriction fragment containing two previously reported 9p breakpoints associated with a alpha-heavy chain disease case (MAL) and KI-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contained the PAX-5 gene which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints of KIS-1 and 1052 were mapped within the 5' noncoding region of PAX-5, while the 9p13 breakpoint of MAL mapped 230 to 270 kb upstream to PAX-5. In all three cases, the translocation caused the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. When compared with six other DLCL cell lines lacking t(9;14)(p13;q32), the KIS-1 cell line showed an 11-fold overexpression of PAX-5 mRNA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. Moreover, metaphase and interphase fluorescence in situ hybridization (FISH) analysis using a YAC clone spanning 1 Mb including the PAX-5 as a probe identified chromosomal translocations in 5 of 7 cases carrying 9p13 translocations. These findings suggest that the PAX-5 gene is the target of the t(9;14) in LPL whereby its expression may be deregulated by juxtaposition to IgH regulatory elements, thus contributing to lymphomagenesis.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

PubMed

Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

2018-05-01

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

Familiar Papillary Thyroid Carcinoma in a Large Brazilian Family Is Not Associated with Succinate Dehydrogenase Defects.

PubMed

Accordi, Elen Dias; Xekouki, Paraskevi; Azevedo, Bruna; de Alexandre, Rodrigo Bertollo; Frasson, Carla; Gantzel, Siliane Marie; Papadakis, Georgios Z; Angelousi, Anna; Stratakis, Constantine A; Sotomaior, Vanessa Santos; Faucz, Fabio R

2016-07-01

Thyroid cancer is the most common endocrine gland malignancy. Advances in understanding the genetic basis for thyroid cancer revealed the potential involvement of several genes in the formation of thyroid tumors. Mutations in the gene coding for succinate dehydrogenase subtype B (SDHB) have been implicated in papillary thyroid cancer (PTC). Succinate dehydrogenase (SDH) is a heterotetrameric protein composed of four subunits, SDHA, SDHB, SDHC, and SDHD, and participates in both the electron transport chain and the tricarboxylic acid cycle. The aim of the study was to evaluate the association between variants in the SDHA, SDHB, SDHC, and SDHD genes and familiar PTC in a large Brazilian family. Four patients with PTC, 1 patient with PTC and gastrointestinal stromal tumor (GIST), 1 patient with GIST, and their relatives - several of them with different thyroid problems - from a large Brazilian family were screened for genetic variations of SDHx genes with the use of polymerase chain reaction-single-stranded conformational polymorphism and direct sequencing. Only one rare variation in SDHA was found in some of the family members, but not segregating with the disease. No other genetic variants of these genes were detected in the family members that presented with PTC and/or GIST. Familiar PTC and a GIST were not associated with SDHx mutations; additional genetic defects, yet unknown, may be responsible for the development of tumor.

Differential Lipid Composition and Gene Expression in the Semi-Russeted “Cox Orange Pippin” Apple Variety

PubMed Central

Legay, Sylvain; Cocco, Emmanuelle; André, Christelle M.; Guignard, Cédric; Hausman, Jean-Francois; Guerriero, Gea

2017-01-01

Russeting is characterized by a particular rough and brown phenotype, which is mainly due to the accumulation of suberin in the inner part of the epidermal cell walls. In our previous bulk transcriptomic analysis, comparing fully russeted, and waxy apple varieties, showed, in apple fruit skin, a massive decreased expression of cutin, wax and some pentacyclic triterpene biosynthesis genes in the russeted varieties, with an expected concomitant enhanced expression of the suberin biosynthetic genes. In the present work, we performed a deep investigation of the aliphatic composition of the cutin, suberin, waxes, and triterpenes in the waxy and russeted patches of the semi-russeted apple variety “Cox Orange Pippin.” A targeted gene expression profiling was performed to validate candidate genes which were identified in our previous work and might be involved in the respective metabolic pathways. Our results showed that a decrease of cuticular waxes, ursolic acid and oleanolic acid, accompanied by an accumulation of alkyl-hydroxycinamates and betulinic acid, occurs in the russeted patches. The suberin monomer composition is characterized by specific occurrence of 20, 22, and 24 carbon aliphatic chains, whereas cutin is mainly represented by common C16 and C18 aliphatic chains. This work depicts, for the first time in apple, the complex composition of suberin, cutin, waxes and triterpenes, and confirms the strong interplay between these epidermal polymers in apple fruit skin. PMID:29018466

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

PubMed Central

Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

2014-01-01

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

Differential Lipid Composition and Gene Expression in the Semi-Russeted "Cox Orange Pippin" Apple Variety.

PubMed

Legay, Sylvain; Cocco, Emmanuelle; André, Christelle M; Guignard, Cédric; Hausman, Jean-Francois; Guerriero, Gea

2017-01-01

Russeting is characterized by a particular rough and brown phenotype, which is mainly due to the accumulation of suberin in the inner part of the epidermal cell walls. In our previous bulk transcriptomic analysis, comparing fully russeted, and waxy apple varieties, showed, in apple fruit skin, a massive decreased expression of cutin, wax and some pentacyclic triterpene biosynthesis genes in the russeted varieties, with an expected concomitant enhanced expression of the suberin biosynthetic genes. In the present work, we performed a deep investigation of the aliphatic composition of the cutin, suberin, waxes, and triterpenes in the waxy and russeted patches of the semi-russeted apple variety "Cox Orange Pippin." A targeted gene expression profiling was performed to validate candidate genes which were identified in our previous work and might be involved in the respective metabolic pathways. Our results showed that a decrease of cuticular waxes, ursolic acid and oleanolic acid, accompanied by an accumulation of alkyl-hydroxycinamates and betulinic acid, occurs in the russeted patches. The suberin monomer composition is characterized by specific occurrence of 20, 22, and 24 carbon aliphatic chains, whereas cutin is mainly represented by common C16 and C18 aliphatic chains. This work depicts, for the first time in apple, the complex composition of suberin, cutin, waxes and triterpenes, and confirms the strong interplay between these epidermal polymers in apple fruit skin.

Genome analysis of Desulfotomaculum gibsoniae strain GrollT a highly versatile Gram-positive sulfate-reducing bacterium

PubMed Central

Kuever, Jan; Visser, Michael; Loeffler, Claudia; Boll, Matthias; Worm, Petra; Sousa, Diana Z.; Plugge, Caroline M.; Schaap, Peter J.; Muyzer, Gerard; Pereira, Ines A.C.; Parshina, Sofiya N.; Goodwin, Lynne A.; Kyrpides, Nikos C.; Detter, Janine; Woyke, Tanja; Chain, Patrick; Davenport, Karen W.; Rohde, Manfred; Spring, Stefan; Klenk, Hans-Peter; Stams, Alfons J.M.

2014-01-01

Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain GrollT together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes. PMID:25197466

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer.

PubMed

Shao, Yaping; Ye, Guozhu; Ren, Shancheng; Piao, Hai-Long; Zhao, Xinjie; Lu, Xin; Wang, Fubo; Ma, Wang; Li, Jia; Yin, Peiyuan; Xia, Tian; Xu, Chuanliang; Yu, Jane J; Sun, Yinghao; Xu, Guowang

2018-07-15

Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies. © 2018 UICC.

Individual and combined influence of ACE and ACTN3 genes on muscle phenotypes in Polish athletes.

PubMed

Orysiak, Joanna; Mazur-Różycka, Joanna; Busko, Krzysztof; Gajewski, Jan; Szczepanska, Beata; Malczewska-Lenczowska, Jadwiga

2017-02-08

The aim of this study was to examine the association between ACE and ACTN3 genes, independently or in combination, and muscle strength and power in male and female athletes. The study involved 398 young male (n=266) and female (n=132) athletes representing various sport disciplines (ice hockey, canoeing, swimming, volleyball). All were Caucasians. The following measurements were taken: height of jump and mechanical power in countermovement jump (CMJ) and spike jump (SPJ), and muscle strength of 10 muscle groups (flexors and extensors of the elbow, shoulder, hip, knee and trunk). The ID polymorphism of ACE and the R577X polymorphism of ACTN3 were typed using PCR (polymerase chain reaction) and PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism), respectively. The genotype distribution of the ACE and ACTN3 genes did not differ significantly between groups of athletes for either sex. There was no association between ACE and ACTN3 genotypes (alone or in combination) and sum of muscle strength, height of jump or mechanical power in both jump tests (CMJ and SPJ) for male and female athletes. These findings do not support an influential role of the ACE and ACTN3 genes in determining power/strength performance of elite athletes.

Effect of medium/ω-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

PubMed Central

Cury-Boaventura, M F; Gorjão, R; Martins de Lima, T; Fiamoncini, J; Godoy, A B P; Deschamphs, F C; Soriano, F G; Curi, R

2011-01-01

Lipid emulsion (LE) containing medium/ω-6 long chain triglyceride-based emulsion (MCT/ω-6 LCT LE) has been recommended in the place of ω-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/ω-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/ω-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/ω-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/ω-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription. PMID:21682721

Basic Helix-Loop-Helix Transcription Factor Bmsage Is Involved in Regulation of fibroin H-chain Gene via Interaction with SGF1 in Bombyx mori

PubMed Central

Li, Qiong-Yan; Hu, Wen-Bo; Zhou, Meng-Ting; Nie, Hong-Yi; Zhang, Yin-Xia; Peng, Zhang-Chuan; Zhao, Ping; Xia, Qing-You

2014-01-01

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix–loop–helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells. PMID:24740008

Functional characterization of an invertase inhibitor gene involved in sucrose metabolism in tomato fruit.

PubMed

Zhang, Ning; Jiang, Jing; Yang, Yan-li; Wang, Zhi-he

2015-10-01

In this study, we produced tomato plants overexpressing an invertase inhibitor gene (Sly-INH) from tomato, using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction (PCR). Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase (CWIN), which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.

SxtA gene sequence analysis of dinoflagellate Alexandrium minutum

NASA Astrophysics Data System (ADS)

Norshaha, Safida Anira; Latib, Norhidayu Abdul; Usup, Gires; Yusof, Nurul Yuziana Mohd

2015-09-01

The dinoflagellate Alexandrium minutum is typically known for the production of potent neurotoxins such as saxitoxin, affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). These phenomena is related to the harmful algal blooms (HABs) that is believed to be influenced by environmental and nutritional factors. Previous study has revealed that SxtA gene is a starting gene that involved in the saxitoxin production pathway. The aim of this study was to analyse the sequence of the sxtA gene in A. minutum. The dinoflagellates culture was cultured at temperature 26°C with 16:8-hour light:dark photocycle. After the samples were harvested, RNA was extracted, complementary DNA (cDNA) was synthesised and amplified by polymerase chain reaction (PCR). The PCR products were then purified and cloned before sequenced. The SxtA sequence obtained was then analyzed in order to identify the presence of SxtA gene in Alexandrium minutum.

Molecular evaluation of five cardiac genes in Doberman Pinschers with dilated cardiomyopathy.

PubMed

Meurs, Kathryn M; Hendrix, Kristina P; Norgard, Michelle M

2008-08-01

To sequence the exonic and splice site regions of 5 cardiac genes associated with the human form of familial dilated cardiomyopathy (DCM) in Doberman Pinschers with DCM and to identify a causative mutation. 5 unrelated Doberman Pinschers with DCM and 2 unaffected Labrador Retrievers (control dogs). Exonic and splice site regions of the 5 genes encoding the cardiac proteins troponin C, lamin A/C, cysteine- and glycine-rich protein 3, cardiac troponin T, and the beta-myosin heavy chain were sequenced. Sequences were compared for nucleotide changes between affected dogs and the published canine sequences and 2 control dogs. Base pair changes were considered to be causative for DCM if they were present in an affected dog but not in the control dogs or published sequences and if they involved a conserved amino acid and changed that amino acid to a different polarity, acid-base status, or structure. A causative mutation for DCM in Doberman Pinschers was not identified, although single nucleotide polymorphisms were detected in some dogs in the cysteine- and glycine-rich protein 3, beta-myosin heavy chain, and troponin T genes. Mutations in 5 of the cardiac genes associated with the development of DCM in humans did not appear to be causative for DCM in Doberman Pinschers. Continued evaluation of additional candidate genes or a focused approach with an association analysis is warranted to elucidate the molecular cause of this important cardiac disease in Doberman Pinschers.

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

PubMed Central

Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing

2012-01-01

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425

Reduced Dermatopontin Expression Is a Molecular Link Between Uterine Leiomyomas and Keloids

PubMed Central

Catherino, William H.; Leppert, Phyllis C.; Stenmark, Matthew H.; Payson, Mark; Potlog-Nahari, Clariss; Nieman, Lynnette K.; Segars, James H.

2014-01-01

Uterine leiomyomas are prevalent estrogen-responsive clonal tumors, but the specific genetic alterations that contribute to their development have not been elucidated. To identify genes involved in the formation of leiomyomas, we used global expression profiling to compare clonal tumors with normal myometrium. Contrary to expectation, genes involved in estrogen action were not differentially expressed between leiomyoma and normal myometrium. Genes encoding extracellular-matrix proteins were prominently featured, suggesting their involvement in formation of a myofibroblast phenotype. Analysis of the extracellular matrix in the leiomyomas revealed a disordered collagen fibril orientation. Expression of the collagen-binding protein dermatopontin was found to be consistently decreased in leiomyoma by both reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR (mean underexpression = 9.41-fold) regardless of leiomyoma size, leiomyoma location, patient race, and patient age. This expression pattern was observed in 11 subjects and a total of 23 leiomyoma: myometrium pairs. Decreased expression of dermatopontin was also associated with keloid formation, a fibrotic disease that shares epidemiologic similarities with leiomyoma. Immunohistochemical studies of leiomyomas and keloids demonstrated reduced levels of dermatopontin in both tissues. In addition, ultrastructural analysis revealed that the orientation of the collagen fibrils in the keloid tissues strongly resembled that in the leiomyomas. Reduction in dermatopontin was associated with an increase in transforming growth factor–β3 (TGFB3) mRNA levels in leiomyomas, whereas other genes involved in dermatopontin signaling were not differentially expressed. These findings suggest that leiomyoma development involves a myofibroblast cell phenotype characterized by dysregulation of genes encoding extracellular-matrix proteins. In particular, decreased expression of dermatopontin represents a molecular link between the leiomyoma and keloid phenotypes. PMID:15139000

A murC gene from coryneform bacteria.

PubMed

Wachi, M; Wijayarathna, C D; Teraoka, H; Nagai, K

1999-02-01

The upstream flanking region of the ftsQ and ftsZ genes of Brevibacterium flavum MJ233, which belongs to the coryneform bacteria, was amplified by the inverse polymerase chain reaction method and cloned in Escherichia coli. Complementation analysis of E. coli mutant with a defective cell-wall synthesis mechanism with the cloned fragment and its DNA sequencing indicated the presence of the murC gene, encoding UDP-N-acetylmuramate:L-alanine ligase involved in peptidoglycan synthesis, just upstream from the ftsQ gene. The B. flavum murC gene could encode a protein of 486 amino acid residues with a calculated molecular mass of 51 198 Da. A 50-kDa protein was synthesized by the B. flavum murC gene in an in vitro transcription/translation system using E. coli S30 lysate. These results indicate that the genes responsible for cell-wall synthesis and cell division are located as a cluster in B. flavum similar to the E. coli mra region.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex.

PubMed

Łopieńska-Biernat, E; Zaobidna, E A; Dmitryjuk, M

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen-trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)-in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

PubMed Central

Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

MAOA and TNF-β gene polymorphisms are associated with photophobia but not osmophobia in patients with migraine.

PubMed

Ishii, Masakazu; Usami, Shino; Hara, Hajime; Imagawa, Atsuko; Masuda, Yutaka; Shimizu, Shuniichi

2014-06-01

Photophobia and osmophobia are typical symptoms associated with migraine, but the contributions of gene polymorphisms to these symptoms are not fully elucidated. We investigated whether the gene polymorphisms are involved in photophobia and osmophobia in patients with migraine. Ninety-one migraine patients and 119 non-headache healthy volunteers were enrolled. The 12 gene polymorphisms were determined by polymerase-chain-reaction (PCR) and PCR restriction-fragment-length polymorphism analysis. Photophobia and osmophobia were observed in 49 (54%) and 31 patients (34%), respectively. Distributions of monoamine oxidase A (MAOA) T941G and tumour necrosis factor-β (TNF-β) G252A polymorphisms were significantly different between patients with photophobia and controls. However, no gene polymorphism differences were observed between patients with osmophobia and controls. The MAOA T941G and TNF-β G252A gene polymorphisms appear to contribute to photophobia but not to osmophobia. We propose that different gene polymorphisms are responsible for photophobia and osmophobia symptoms during migraine.

Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

PubMed

Bolland, Daniel J; Wood, Andrew L; Corcoran, Anne E

2009-01-01

V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region.

PubMed

Wozniak, K; Piaskowski, S; Gresner, S M; Golanska, E; Bieniek, E; Bigoszewska, K; Sikorska, B; Szybka, M; Kulczycka-Wojdala, D; Zakrzewska, M; Zawlik, I; Papierz, W; Stawski, R; Jaskolski, D J; Och, W; Sieruta, M; Liberski, P P; Rieske, P

2008-05-01

Neurofibromin 2 (NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), alpha-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH (P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

Molecular biology of myopia.

PubMed

Schaeffel, Frank; Simon, Perikles; Feldkaemper, Marita; Ohngemach, Sibylle; Williams, Robert W

2003-09-01

Experiments in animal models of myopia have emphasised the importance of visual input in emmetropisation but it is also evident that the development of human myopia is influenced to some degree by genetic factors. Molecular genetic approaches can help to identify both the genes involved in the control of ocular development and the potential targets for pharmacological intervention. This review covers a variety of techniques that are being used to study the molecular biology of myopia. In the first part, we describe techniques used to analyse visually induced changes in gene expression: Northern Blot, polymerase chain reaction (PCR) and real-time PCR to obtain semi-quantitative and quantitative measures of changes in transcription level of a known gene, differential display reverse transcription PCR (DD-RT-PCR) to search for new genes that are controlled by visual input, rapid amplification of 5' cDNA (5'-RACE) to extend the 5' end of sequences that are regulated by visual input, in situ hybridisation to localise the expression of a given gene in a tissue and oligonucleotide microarray assays to simultaneously test visually induced changes in thousands of transcripts in single experiments. In the second part, we describe techniques that are used to localise regions in the genome that contain genes that are involved in the control of eye growth and refractive errors in mice and humans. These include quantitative trait loci (QTL) mapping, exploiting experimental test crosses of mice and transmission disequilibrium tests (TDT) in humans to find chromosomal intervals that harbour genes involved in myopia development. We review several successful applications of this battery of techniques in myopia research.

Multifocal Epithelial Hyperplasia of Oral Cavity Expressing HPV 16 Gene: A Rare Entity

PubMed Central

Prabhat, M. P. V.; Raja Lakshmi, Chintamaneni; Sai Madhavi, N.; Bhavana, Sujana Mulk; Sarat, Gummadapu; Ramamohan, Kodali

2013-01-01

Focal epithelial hyperplasia is a rare contagious disease caused by human papilloma virus. Usually HPV involves either cutaneous or mucosal surfaces, whereas concomitant mucocutaneous involvement is extremely rare. We report such a unique case of multifocal epithelial hyperplasia involving multiple sites of oral cavity along with skin lesions in a 65-year-old female. We also discuss the probable multifactorial etiology and variable clinical presentations of the lesions, including evidence of HPV 16 expression, as detected by polymerase chain reaction. The present report illustrates the need for careful examination and prompt diagnosis of the disease, as it might be associated with high risk genotypes such as HPV 16 and 18. PMID:24455323

Blood-induced differential gene expression in Anopheles dirus evaluated using RNA sequencing.

PubMed

Mongkol, W; Nguitragool, W; Sattabongkot, J; Kubera, A

2018-06-08

Malaria parasites are transmitted through blood feeding by female Anopheline mosquitoes. Unveiling the blood-feeding process will improve understanding of vector biology. Anopheles dirus (Diptera: Culicidae) is one of the primary malaria vectors in the Greater Mekong Subregion, the epicentre of malaria drug resistance. In this study, differential gene expression between sugar- and blood-fed An. dirus was investigated by RNA sequencing (RNA-seq). A total of 589 transcripts were found to be upregulated and 703 transcripts downregulated as a result of blood feeding. Transcriptional differences were found in genes involved in blood digestion, peritrophic matrix formation, oogenesis and vitellogenesis. The expression levels of several genes were validated by quantitative reverse transcription polymerase chain reaction. The present results provide better understanding of An. dirus biology in relation to its blood feeding. © 2018 The Royal Entomological Society.

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

PubMed

Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

2003-08-14

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor.

PubMed

Bernard, Clémence; Vincent, Clémentine; Testa, Damien; Bertini, Eva; Ribot, Jérôme; Di Nardo, Ariel A; Volovitch, Michel; Prochiantz, Alain

2016-05-01

During postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells). In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these "transfer" sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity. We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system.

Correlations between FAS elongation cycle genes expression and fatty acid production for improvement of long-chain fatty acids in Escherichia coli.

PubMed

Lee, Sunhee; Jung, Yeontae; Lee, Seunghan; Lee, Jinwon

2013-03-01

Microorganisms have been used for biodiesel (fatty acid methyl ester) production due to their significant environmental and economic benefits. The aim of the present research was to develop new strains of Escherichia coli K-12 MG1655 and to increase the content of long-chain fatty acids by overexpressing essential enzymes that are involved in the fatty acid synthase elongation cycle. In addition, the relationship of β-ketoacyl-acyl carrier protein (ACP) synthase (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydrase (fabZ), and β-enoyl-ACP reductase (fabI) with respect to fatty acid production was investigated. The four enzymes play a unique role in fatty acid biosynthesis and elongation processes. We report the generation of recombinant E. coli strains that produced long-chain fatty acids to amounts twofold over wild type. To verify the results, NAD(+)/NADH ratios and glucose analyses were performed. We also confirmed that FabZ plays an important role in producing unsaturated fatty acids (UFAs) as E. coli SGJS25 (overexpressing the fabZ gene) produced the highest percentage of UFAs (35 % of total long-chain fatty acids), over wild type and other recombinants. Indeed, cis-9-hexadecenoic acid, a major UFA in E. coli SGJS25, was produced at levels 20-fold higher than in wild type after 20 h in culture. The biochemically engineered E. coli presented in this study is expected to be more economical for producing long-chain fatty acids in quality biodiesel production processes.

Lipid metabolism in Rhodnius prolixus: Lessons from the genome.

PubMed

Majerowicz, David; Calderón-Fernández, Gustavo M; Alves-Bezerra, Michele; De Paula, Iron F; Cardoso, Lívia S; Juárez, M Patricia; Atella, Georgia C; Gondim, Katia C

2017-01-05

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from β-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid β-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro-aerobic conditions

PubMed Central

Liu, Jingjing; Sun, Faqian; Wang, Liang; Ju, Xi; Wu, Weixiang; Chen, Yingxu

2014-01-01

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro-aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real-time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro-aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD. PMID:24245852

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

PubMed

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell.

PubMed

Kumar, Rashmi; Bach, Martina P; Mainoldi, Federica; Maruya, Mikako; Kishigami, Satoshi; Jumaa, Hassan; Wakayama, Teruhiko; Kanagawa, Osami; Fagarasan, Sidonia; Casola, Stefano

2015-02-03

In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(NT) animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.

Infrared microscopic imaging of cutaneous wound healing: lipid conformation in the migrating epithelial tongue

NASA Astrophysics Data System (ADS)

Yu, Guo; Stojadinovic, Olivera; Tomic-Canic, Marjana; Flach, Carol R.; Mendelsohn, Richard

2012-09-01

Infrared microscopic imaging has been utilized to analyze for the first time the spatial distribution of lipid structure in an ex vivo human organ culture skin wound healing model. Infrared images were collected at zero, two, four, and six days following wounding. Analysis of lipid infrared spectral properties revealed the presence of a lipid class with disordered chains within and in the vicinity of the migrating epithelial tongue. The presence of lipid ester C=O bands colocalized with the disordered chains provided evidence for the presence of carbonyl-containing lipid species. Gene array data complemented the biophysical studies and provided a biological rationale for the generation of the disordered chain species. This is the first clear observation, to our knowledge, of disordered lipid involvement in cutaneous wound healing. Several possibilities are discussed for the biological relevance of these observations.

Lack of association between atopic eczema and the genetic variants of interleukin-4 and the interleukin-4 receptor alpha chain gene: heterogeneity of genetic backgrounds on immunoglobulin E production in atopic eczema patients.

PubMed

Tanaka, K; Sugiura, H; Uehara, M; Hashimoto, Y; Donnelly, C; Montgomery, D S

2001-10-01

The genetic background of atopic eczema might be heterogeneous and there is a possibility that immunoglobulin (Ig)E responsiveness in patients with atopic eczema is controlled separately from the development of atopic eczema. Although both interleukin (IL)-4 and the IL-4 receptor alpha chain have an important role for IgE production and are therefore possible candidate genes for atopy, it has not been clarified whether these genes play any roles in atopic eczema patients who have normal IgE productivity. We aimed to assess whether the polymorphisms of the IL-4 gene and the IL-4 receptor alpha chain gene play any roles in atopic eczema patients, particularly in patients who have normal IgE productivity. We determined the genotype with regard to polymorphisms in the genes for IL-4 and the IL-4 receptor alpha chain (- 589C/T of IL-4; Ile50Val, Ala375Glu and Arg551Gln of IL-4 receptor alpha chain) in patients with atopic eczema using the fluorogenic 5' nuclease assay. IL-4 and the IL-4 receptor alpha chain genotypes were not significantly associated with either total patients with atopic eczema or atopic eczema patients who had normal IgE productivity. The distribution of genotypes of IL-4-589C/T differed by the serum IgE levels in patients with atopic eczema. These results suggest that the polymorphisms in the IL-4 gene and the IL-4 receptor alpha chain gene play no role in the development of atopic eczema in patients who have normal IgE productivity.

Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

PubMed Central

Liljavirta, Jenni; Niku, Mikael; Pessa-Morikawa, Tiina; Ekman, Anna; Iivanainen, Antti

2014-01-01

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period. PMID:24926997

Promoter methylation profile in gallbladder cancer.

PubMed

Roa, Juan Carlos; Anabalón, Leonardo; Roa, Iván; Melo, Angélica; Araya, Juan Carlos; Tapia, Oscar; de Aretxabala, Xavier; Muñoz, Sergio; Schneider, Barbara

2006-03-01

Methylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time. Twenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained. All cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied. The high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

PubMed

Morona, R; van den Bosch, L; Manning, P A

1995-02-01

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).

Cardiac transcriptional response to acute and chronic angiotensin II treatments.

PubMed

Larkin, Jennie E; Frank, Bryan C; Gaspard, Renee M; Duka, Irena; Gavras, Haralambos; Quackenbush, John

2004-07-08

Exposure of experimental animals to increased angiotensin II (ANG II) induces hypertension associated with cardiac hypertrophy, inflammation, and myocardial necrosis and fibrosis. Some of the most effective antihypertensive treatments are those that antagonize ANG II. We investigated cardiac gene expression in response to acute (24 h) and chronic (14 day) infusion of ANG II in mice; 24-h treatment induces hypertension, and 14-day treatment induces hypertension and extensive cardiac hypertrophy and necrosis. For genes differentially expressed in response to ANG II treatment, we tested for significant regulation of pathways, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Microarray Pathway Profiler (GenMAPP) databases, as well as functional classes based on Gene Ontology (GO) terms. Both acute and chronic ANG II treatments resulted in decreased expression of mitochondrial metabolic genes, notably those for the electron transport chain and Krebs-TCA cycle; chronic ANG II treatment also resulted in decreased expression of genes involved in fatty acid metabolism. In contrast, genes involved in protein translation and ribosomal activity increased expression following both acute and chronic ANG II treatments. Some classes of genes showed differential response between acute and chronic ANG II treatments. Acute treatment increased expression of genes involved in oxidative stress and amino acid metabolism, whereas chronic treatments increased cytoskeletal and extracellular matrix genes, second messenger cascades responsive to ANG II, and amyloidosis genes. Although a functional linkage between Alzheimer disease, hypertension, and high cholesterol has been previously documented in studies of brain tissue, this is the first demonstration of induction of Alzheimer disease pathways by hypertension in heart tissue. This study provides the most comprehensive available survey of gene expression changes in response to acute and chronic ANG II treatment, verifying results from disparate studies, and suggests mechanisms that provide novel insight into the etiology of hypertensive heart disease and possible therapeutic interventions that may help to mitigate its effects.

Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma

PubMed Central

Berget, Ellen; Helgeland, Lars; Liseth, Knut; Løkeland, Turid; Molven, Anders; Vintermyr, Olav Karsten

2014-01-01

Aims We aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL). Methods Clonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis. Results BM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I–III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0–1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival. Conclusions Our results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor. PMID:25233852

Primary and secondary CoQ(10) deficiencies in humans.

PubMed

Quinzii, Catarina M; Hirano, Michio

2011-01-01

CoQ(10) deficiencies are clinically and genetically heterogeneous. This syndrome has been associated with five major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) cerebellar ataxia, (4) isolated myopathy, and (5) nephrotic syndrome. In a few patients, pathogenic mutations have been identified in genes involved in the biosynthesis of CoQ(10) (primary CoQ(10) deficiencies) or in genes not directly related to CoQ(10) biosynthesis (secondary CoQ(10) deficiencies). Respiratory chain defects, ROS production, and apoptosis variably contribute to the pathogenesis of primary CoQ(10) deficiencies. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.

2000-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.

2001-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

PubMed

Fernández, Ignacio; Darias, Maria; Andree, Karl B; Mazurais, David; Zambonino-Infante, Jose Luís; Gisbert, Enric

2011-02-09

Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

PubMed Central

2011-01-01

Background Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. Results During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Conclusions Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages. PMID:21306609

The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

PubMed Central

Santiago, Brendaliz; MacGilvray, Matthew; Faustoferri, Roberta C.

2012-01-01

The ability of Streptococcus mutans to produce and tolerate organic acids from carbohydrate metabolism represents a major virulence factor responsible for the formation of carious lesions. Pyruvate is a key metabolic intermediate that, when rerouted to other metabolic pathways such as amino acid biosynthesis, results in the alleviation of acid stress by reducing acid end products and aiding in maintenance of intracellular pH. Amino acid biosynthetic genes such as ilvC and ilvE were identified as being upregulated in a proteome analysis of Streptococcus mutans under acid stress conditions (A. C. Len, D. W. Harty, and N. A. Jacques, Microbiology 150:1353–1366, 2004). In Lactococcus lactis and Staphylococcus carnosus, the ilvE gene product is involved with biosynthesis and degradation of branched-chain amino acids, as well as in the production of branched-chain fatty acids (B. Ganesan and B. C. Weimer, Appl. Environ. Microbiol. 70:638–641, 2004; S. M. Madsen et al., Appl. Environ. Microbiol. 68:4007–4014, 2002; and M. Yvon, S. Thirouin, L. Rijnen, D. Fromentier, and J. C. Gripon, Appl. Environ. Microbiol. 63:414–419, 1997). Here we constructed and characterized an ilvE deletion mutant of S. mutans UA159. Growth experiments revealed that the ilvE mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of ilvE causes a decrease in acid tolerance. The ilvE strain exhibits a defect in F1-Fo ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the ilvE promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acid-adaptive repertoire of S. mutans. PMID:22328677

Ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods.

PubMed

Zhao, Yaofeng; Cui, Huiting; Whittington, Camilla M; Wei, Zhiguo; Zhang, Xiaofeng; Zhang, Ziding; Yu, Li; Ren, Liming; Hu, Xiaoxiang; Zhang, Yaping; Hellman, Lars; Belov, Katherine; Li, Ning; Hammarström, Lennart

2009-09-01

The evolutionary origins of mammalian immunoglobulin H chain isotypes (IgM, IgD, IgG, IgE, and IgA) are still incompletely understood as these isotypes differ considerably in structure and number from their counterparts in nonmammalian tetrapods. We report in this study that the platypus (Ornithorhynchus anatinus) Ig H chain constant region gene locus contains eight Ig encoding genes, which are arranged in an mu-delta-omicron-gamma2-gamma1-alpha1-epsilon-alpha2 order, spanning a total of approximately 200 kb DNA, encoding six distinct isotypes. The omicron (omicron for Ornithorhynchus) gene encodes a novel Ig H chain isotype that consists of four constant region domains and a hinge, and is structurally different from any of the five known mammalian Ig classes. This gene is phylogenetically related to upsilon (epsilon) and gamma, and thus appears to be a structural intermediate between these two genes. The platypus delta gene encodes ten heavy chain constant region domains, lacks a hinge region and is similar to IgD in amphibians and fish, but strikingly different from that in eutherian mammals. The platypus Ig H chain isotype repertoire thus shows a unique combination of genes that share similarity both to those of nonmammalian tetrapods and eutherian animals and demonstrates how phylogenetically informative species can be used to reconstruct the evolutionary history of functionally important genes.

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

PubMed

Nascimento, Diana S; do Vale, Ana; Tomás, Ana M; Zou, Jun; Secombes, Christopher J; dos Santos, Nuno M S

2007-03-01

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Transcriptomic analysis reveals metabolic switches and surface remodeling as key processes for stage transition in Trypanosoma cruzi

PubMed Central

Greif, Gonzalo; Rodriguez, Matias; Alvarez-Valin, Fernando

2017-01-01

American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions. PMID:28286708

Oil palm phenolics confer neuroprotective effects involving cognitive and motor functions in mice

PubMed Central

Leow, Soon-Sen; Sekaran, Shamala Devi; Tan, YewAi; Sundram, Kalyana; Sambanthamurthi, Ravigadevi

2013-01-01

Objectives Phenolics are important phytochemicals which have positive effects on chronic diseases, including neurodegenerative ailments. The oil palm (Elaeis guineensis) is a rich source of water-soluble phenolics. This study was carried out to discover the effects of administering oil palm phenolics (OPP) to mice, with the aim of identifying whether these compounds possess significant neuroprotective properties. Methods OPP was given to BALB/c mice on a normal diet as fluids for 6 weeks while the controls were given distilled water. These animals were tested in a water maze and on a rotarod weekly to assess the effects of OPP on cognitive and motor functions, respectively. Using Illumina microarrays, we further explored the brain gene expression changes caused by OPP in order to determine the molecular mechanisms involved. Real-time quantitative reverse transcription-polymerase chain reaction experiments were then carried out to validate the microarray data. Results We found that mice given OPP showed better cognitive function and spatial learning when tested in a water maze, and their performance also improved when tested on a rotarod, possibly due to better motor function and balance. Microarray gene expression analysis showed that these compounds up-regulated genes involved in brain development and activity, such as those under the regulation of the brain-derived neurotrophic factor. OPP also down-regulated genes involved in inflammation. Discussion These results suggest that the improvement of mouse cognitive and motor functions by OPP is caused by the neuroprotective and anti-inflammatory effects of the extract. PMID:23433062

Regulation Mechanism Mediated by Trans-Encoded sRNA Nc117 in Short Chain Alcohols Tolerance in Synechocystis sp. PCC 6803.

PubMed

Bi, Yanqi; Pei, Guangsheng; Sun, Tao; Chen, Zixi; Chen, Lei; Zhang, Weiwen

2018-01-01

Microbial small RNAs (sRNAs) play essential roles against many stress conditions in cyanobacteria. However, little is known on their regulatory mechanisms on biofuels tolerance. In our previous sRNA analysis, a trans -encoded sRNA Nc117 was found involved in the tolerance to ethanol and 1-butanol in Synechocystis sp. PCC 6803. However, its functional mechanism is yet to be determined. In this study, functional characterization of sRNA Nc117 was performed. Briefly, the exact length of the trans -encoded sRNA Nc117 was determined to be 102 nucleotides using 3' RACE, and the positive regulation of Nc117 on short chain alcohols tolerance was further confirmed. Then, computational target prediction and transcriptomic analysis were integrated to explore the potential targets of Nc117. A total of 119 up-regulated and 116 down-regulated genes were identified in nc117 overexpression strain compared with the wild type by comparative transcriptomic analysis, among which the upstream regions of five genes were overlapped with those predicted by computational target approach. Based on the phenotype analysis of gene deletion and overexpression strains under short chain alcohols stress, one gene slr0007 encoding D-glycero-alpha-D-manno-heptose 1-phosphate guanylyltransferase was determined as a potential target of Nc117, suggesting that the synthesis of LPS or S-layer glycoprotein may be responsible for the tolerance enhancement. As the first reported trans -encoded sRNA positively regulating biofuels tolerance in cyanobacteria, this study not only provided evidence for a new regulatory mechanism of trans -encoded sRNA in cyanobacteria, but also valuable information for rational construction of high-tolerant cyanobacterial chassis.

Mechanisms of neuroimmune gene induction in alcoholism.

PubMed

Crews, Fulton T; Vetreno, Ryan P

2016-05-01

Alcoholism is a primary, chronic relapsing disease of brain reward, motivation, memory, and related circuitry. It is characterized by an individual's continued drinking despite negative consequences related to alcohol use, which is exemplified by alcohol use leading to clinically significant impairment or distress. Chronic alcohol consumption increases the expression of innate immune signaling molecules (ISMs) in the brain that alter cognitive processes and promote alcohol drinking. Unraveling the mechanisms of alcohol-induced neuroimmune gene induction is complicated by positive loops of multiple cytokines and other signaling molecules that converge on nuclear factor kappa-light-chain-enhancer of activated B cells and activator protein-1 leading to induction of additional neuroimmune signaling molecules that amplify and expand the expression of ISMs. Studies from our laboratory employing reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA, immunohistochemistry and Western blot analysis to assess protein expression, and others suggest that ethanol increases brain neuroimmune gene and protein expression through two distinct mechanisms involving (1) systemic induction of innate immune molecules that are transported from blood to the brain and (2) the direct release of high-mobility group box 1 (HMGB1) from neurons in the brain. Released HMGB1 signals through multiple receptors, particularly Toll-like receptor (TLR) 4, that potentiate cytokine receptor responses leading to a hyperexcitable state that disrupts neuronal networks and increases excitotoxic neuronal death. Innate immune gene activation in brain is persistent, consistent with the chronic relapsing disease that is alcoholism. Expression of HMGB1, TLRs, and other ISMs is increased several-fold in the human orbital frontal cortex, and expression of these molecules is highly correlated with each other as well as lifetime alcohol consumption and age of drinking onset. The persistent and cumulative nature of alcohol on HMGB1 and TLR gene induction support their involvement in alcohol-induced long-term changes in brain function and neurodegeneration.

Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

PubMed

Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

2015-07-01

Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

Deletion of the transcriptional coactivator PGC1α in skeletal muscles is associated with reduced expression of genes related to oxidative muscle function

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

Hatazawa, Yukino; Research Fellow of Japan Society for the Promotion of Science, Tokyo; Minami, Kimiko

The expression of the transcriptional coactivator PGC1α is increased in skeletal muscles during exercise. Previously, we showed that increased PGC1α leads to prolonged exercise performance (the duration for which running can be continued) and, at the same time, increases the expression of branched-chain amino acid (BCAA) metabolism-related enzymes and genes that are involved in supplying substrates for the TCA cycle. We recently created mice with PGC1α knockout specifically in the skeletal muscles (PGC1α KO mice), which show decreased mitochondrial content. In this study, global gene expression (microarray) analysis was performed in the skeletal muscles of PGC1α KO mice compared withmore » that of wild-type control mice. As a result, decreased expression of genes involved in the TCA cycle, oxidative phosphorylation, and BCAA metabolism were observed. Compared with previously obtained microarray data on PGC1α-overexpressing transgenic mice, each gene showed the completely opposite direction of expression change. Bioinformatic analysis of the promoter region of genes with decreased expression in PGC1α KO mice predicted the involvement of several transcription factors, including a nuclear receptor, ERR, in their regulation. As PGC1α KO microarray data in this study show opposing findings to the PGC1α transgenic data, a loss-of-function experiment, as well as a gain-of-function experiment, revealed PGC1α’s function in the oxidative energy metabolism of skeletal muscles. - Highlights: • Microarray analysis was performed in the skeletal muscle of PGC1α KO mice. • Expression of genes in the oxidative energy metabolism was decreased. • Bioinformatic analysis of promoter region of the genes predicted involvement of ERR. • PGC1α KO microarray data in this study show the mirror image of transgenic data.« less

Emotional disorders induced by Hemopressin and RVD-hemopressin(α) administration in rats.

PubMed

Leone, Sheila; Recinella, Lucia; Chiavaroli, Annalisa; Martinotti, Sara; Ferrante, Claudio; Mollica, Adriano; Macedonio, Giorgia; Stefanucci, Azzurra; Dvorácskó, Szabolcs; Tömböly, Csaba; De Petrocellis, Luciano; Vacca, Michele; Brunetti, Luigi; Orlando, Giustino

2017-12-01

The endocannabinoid (eCB) system plays an important role in regulating emotional disorders, and is involved, directly or indirectly, in psychiatric diseases, such as anxiety and depression. Hemopressin, a hemoglobin α chain-derived peptide, and RVD-hemopressin(α), a N-terminally extended form of hemopressin, act as antagonist/inverse agonist and negative allosteric modulator of the cannabinoid 1 (CB1) receptor, respectively. Considering the possible involvement of these peptides on emotional behaviour, the aim of our study was to investigate the behavioural effects of a single intraperitoneal (ip) injection of hemopressin (0.05mg/kg) and RVD-hemopressin(α) (0.05mg/kg), using a series of validated behavioural tests (locomotor activity/open field test, light-dark exploration test, forced swim test) in rats. Prefrontal cortex levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and the gene expression of monoamine oxidase (MAO-B) and catechol-O-methyltransferase (COMT) were measured by high performance liquid chromatography (HPLC) analysis and real-time reverse transcription polymerase chain reaction (RT-PCR), respectively. Hemopressin administration induced anxiogenic and depressive behaviour, decreased monoamine steady state levels in prefrontal cortex, and increased the gene expression of the enzymes involved in their catabolism. By contrast, RVD- hemopressin(α) induced anxiolytic and antidepressive effects, increased monoamines and decreased the enzymes in prefrontal cortex. In conclusion, in the present study we demonstrated behavioral effects induced by peripheral hemopressin and RVD-hemopressin(α) injections, that could involve modulatory effects on monoaminergic signaling, in the prefrontal cortex. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma.

PubMed

Tamura, A; Miura, I; Iida, S; Yokota, S; Horiike, S; Nishida, K; Fujii, H; Nakamura, S; Seto, M; Ueda, R; Taniwaki, M

2001-08-01

To detect immunoglobulin heavy chain (IGH) gene translocations with specific oncogene loci, we established an interphase cytogenetic approach using double-color fluorescence in situ hybridization (DC-FISH), which we used to analyze 173 patients with B-cell lymphoma. DC-FISH using the IGH gene (14q32.3) in combination with c-MYC (8q24.1), BCL1 (11q13.3), BCL2 (18q21.3), BCL6 (3q27), and PAX-5 (9p13) gene probes detected IGH translocations in 70 (40.5%) of 173 patients. The partner genes involved in IGH translocations were identified in 56 (80%) of 70 patients, and fusion of the IGH gene with specific oncogenes was detected in 53 of 56 patients, particularly in interphase nuclei of 28 patients for whom cytogenetic analysis was not informative. The most common partner gene was BCL2 (19 patients; 27% of IGH translocation-positive patients), followed by BCL6 (16; 23%), BCL1 (11; 16%), c-MYC (7; 10%), and PAX-5 (2; 3%). These oncogenes were closely associated with subtypes of B-cell lymphoma. The other partners were 19q13 (BCL3), 6p25 (MUM1/IRF4), 1q36, and chromosome 8 identified in one patient each. Six of the nine patients with add(14)(q32) showed a BCL6/IGH translocation. Double translocations of the IGH gene were found in three patients; c-MYC+BCL1, c-MYC+BCL2, and c-MYC+BCL6 in each one. Interphase FISH using specific IGH-translocation probes is valuable for defining clinically meaningful subgroups of B-cell lymphoma.

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

Popp, R.A.; Enlow, M.K.

The clinical hematologic change in 2 groups of progeny from mice carrying radiation-induced strain SEC ..cap alpha..-chain deficiencies was found to be similar to the hematologic alterations in persons with ..cap alpha..-thalassemia. The heterozygous deletion or inactivation of the ..cap alpha..-chain gene in mice caused an anemia similar to ..cap alpha..-thalassemina minor in persons. The ..cap alpha..-chain deficiency in mice created an erythrocytosis, reticulocytosis, and microcytic, hypochromic anemia comparable with the changes in human ..cap alpha..-thalassemia minor resulting from deletion of the ..cap alpha..-chain gene. These mouse mutants are the only known animal models of human thalassemia. A comparison ofmore » hematologic values obtained from progeny possessing an ..cap alpha..-chain gene deficiency and from progeny possessing a ..beta..-chain duplication suggested that the deficiency of ..cap alpha..-chain synthesis, rather than a simple imbalance between the amounts of ..cap alpha..- and ..beta..-chains produced, was primarily responsible for the altered hematologic characteristics in these ..cap alpha..-thalassemic mice.« less

Expression of fox-related genes in the skin follicles of Inner Mongolia cashmere goat.

PubMed

Han, Wenjing; Li, Xiaoyan; Wang, Lele; Wang, Honghao; Yang, Kun; Wang, Zhixin; Wang, Ruijun; Su, Rui; Liu, Zhihong; Zhao, Yanhong; Zhang, Yanjun; Li, Jinquan

2018-03-01

This study investigated the expression of genes in cashmere goats at different periods of their fetal development. Bioinformatics analysis was used to evaluate data obtained by transcriptome sequencing of fetus skin samples collected from Inner Mongolia cashmere goats on days 45, 55, and 65 of fetal age. We found that FoxN1 , FoxE1 , and FoxI3 genes of the Fox gene family were probably involved in the growth and development of the follicle and the formation of hair, which is consistent with previous findings. Real-time quantitative polymerase chain reaction detecting system and Western blot analysis were employed to study the relative differentially expressed genes FoxN1 , FoxE1 , and FoxI3 in the body skin of cashmere goat fetuses and adult individuals. This study provided new fundamental information for further investigation of the genes related to follicle development and exploration of their roles in hair follicle initiation, growth, and development.

Molecular regulation of santalol biosynthesis in Santalum album L.

PubMed

Rani, Arti; Ravikumar, Puja; Reddy, Manjunatha Damodara; Kush, Anil

2013-09-25

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 - a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 - an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis. © 2013.

Identification of the Actinobacillus pleuropneumoniae Leucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes▿

PubMed Central

Wagner, Trevor K.; Mulks, Martha H.

2007-01-01

Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset of A. pleuropneumoniae genes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown in Escherichia coli to regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized that A. pleuropneumoniae contains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of an A. pleuropneumoniae serotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purified A. pleuropneumoniae His6-Lrp binds in vitro to the A. pleuropneumoniae promoter regions for ilvI, antisense cps1AB, lrp, and nqr. A genetically defined A. pleuropneumoniae lrp mutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from the ilvI and antisense cps1AB promoters in wild-type, lrp mutant, and complemented lrp mutant strains indicated that Lrp is required for induction of expression of ilvI under BCAA limitation. PMID:17060463

Neuropathologic Characterization of Pontocerebellar Hypoplasia Type 6 Associated With Cardiomyopathy and Hydrops Fetalis and Severe Multisystem Respiratory Chain Deficiency due to Novel RARS2 Mutations.

PubMed

Lax, Nichola Z; Alston, Charlotte L; Schon, Katherine; Park, Soo-Mi; Krishnakumar, Deepa; He, Langping; Falkous, Gavin; Ogilvy-Stuart, Amanda; Lees, Christoph; King, Rosalind H; Hargreaves, Iain P; Brown, Garry K; McFarland, Robert; Dean, Andrew F; Taylor, Robert W

2015-07-01

Autosomal recessive mutations in the RARS2 gene encoding the mitochondrial arginyl-transfer RNA synthetase cause infantile-onset myoencephalopathy pontocerebellar hypoplasia type 6 (PCH6). We describe 2 sisters with novel compound heterozygous RARS2 mutations who presented perinatally with neurologic features typical of PCH6 but with additional features including cardiomyopathy, hydrops, and pulmonary hypoplasia and who died at 1 day and 14 days of age. Magnetic resonance imaging findings included marked cerebellar hypoplasia, gyral immaturity, punctate lesions in cerebral white matter, and unfused deep cerebral grey matter. Enzyme histochemistry of postmortem tissues revealed a near-global cytochrome c oxidase-deficiency; assessment of respiratory chain enzyme activities confirmed severe deficiencies involving complexes I, III, and IV. Molecular genetic studies revealed 2 RARS2 gene mutations: a c.1A>G, p.? variant predicted to abolish the initiator methionine, and a deep intronic c.613-3927C>T variant causing skipping of exons 6-8 in the mature RARS2 transcript. Neuropathologic investigation included low brain weights, small brainstem and cerebellum, deep cerebral white matter pathology, pontine nucleus neuron loss (in 1 sibling), and peripheral nerve pathology. Mitochondrial respiratory chain immunohistochemistry in brain tissues confirmed an absence of complexes I and IV immunoreactivity with sparing of mitochondrial numbers. These cases expand the clinical spectrum of RARS2 mutations, including antenatal features and widespread mitochondrial respiratory chain deficiencies in postmortem brain tissues.

Neuropathologic Characterization of Pontocerebellar Hypoplasia Type 6 Associated With Cardiomyopathy and Hydrops Fetalis and Severe Multisystem Respiratory Chain Deficiency due to Novel RARS2 Mutations

PubMed Central

Lax, Nichola Z.; Alston, Charlotte L.; Schon, Katherine; Park, Soo-Mi; Krishnakumar, Deepa; He, Langping; Falkous, Gavin; Ogilvy-Stuart, Amanda; Lees, Christoph; King, Rosalind H.; Hargreaves, Iain P.; Brown, Garry K.; McFarland, Robert; Dean, Andrew F.; Taylor, Robert W.

2015-01-01

Abstract Autosomal recessive mutations in the RARS2 gene encoding the mitochondrial arginyl-transfer RNA synthetase cause infantile-onset myoencephalopathy pontocerebellar hypoplasia type 6 (PCH6). We describe 2 sisters with novel compound heterozygous RARS2 mutations who presented perinatally with neurologic features typical of PCH6 but with additional features including cardiomyopathy, hydrops, and pulmonary hypoplasia and who died at 1 day and 14 days of age. Magnetic resonance imaging findings included marked cerebellar hypoplasia, gyral immaturity, punctate lesions in cerebral white matter, and unfused deep cerebral grey matter. Enzyme histochemistry of postmortem tissues revealed a near-global cytochrome c oxidase-deficiency; assessment of respiratory chain enzyme activities confirmed severe deficiencies involving complexes I, III, and IV. Molecular genetic studies revealed 2 RARS2 gene mutations: a c.1A>G, p.? variant predicted to abolish the initiator methionine, and a deep intronic c.613-3927C>T variant causing skipping of exons 6–8 in the mature RARS2 transcript. Neuropathologic investigation included low brain weights, small brainstem and cerebellum, deep cerebral white matter pathology, pontine nucleus neuron loss (in 1 sibling), and peripheral nerve pathology. Mitochondrial respiratory chain immunohistochemistry in brain tissues confirmed an absence of complexes I and IV immunoreactivity with sparing of mitochondrial numbers. These cases expand the clinical spectrum of RARS2 mutations, including antenatal features and widespread mitochondrial respiratory chain deficiencies in postmortem brain tissues. PMID:26083569

Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides.

PubMed

Yang, L-C; Tsai, C-H; Huang, F-M; Su, Y-F; Lai, C-C; Liu, C-M; Chang, Y-C

2004-09-01

To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P < 0.05). In addition, the expression of VEGF depended on the bacteria tested. Black-pigmented Bacteroides may be involved in developing pulpal disease through the stimulation of VEGF production that would lead to the expansion of the vascular network coincident to progression of the inflammation.

Deletion of the transcriptional coactivator PGC1α in skeletal muscles is associated with reduced expression of genes related to oxidative muscle function.

PubMed

Hatazawa, Yukino; Minami, Kimiko; Yoshimura, Ryoji; Onishi, Takumi; Manio, Mark Christian; Inoue, Kazuo; Sawada, Naoki; Suzuki, Osamu; Miura, Shinji; Kamei, Yasutomi

2016-12-09

The expression of the transcriptional coactivator PGC1α is increased in skeletal muscles during exercise. Previously, we showed that increased PGC1α leads to prolonged exercise performance (the duration for which running can be continued) and, at the same time, increases the expression of branched-chain amino acid (BCAA) metabolism-related enzymes and genes that are involved in supplying substrates for the TCA cycle. We recently created mice with PGC1α knockout specifically in the skeletal muscles (PGC1α KO mice), which show decreased mitochondrial content. In this study, global gene expression (microarray) analysis was performed in the skeletal muscles of PGC1α KO mice compared with that of wild-type control mice. As a result, decreased expression of genes involved in the TCA cycle, oxidative phosphorylation, and BCAA metabolism were observed. Compared with previously obtained microarray data on PGC1α-overexpressing transgenic mice, each gene showed the completely opposite direction of expression change. Bioinformatic analysis of the promoter region of genes with decreased expression in PGC1α KO mice predicted the involvement of several transcription factors, including a nuclear receptor, ERR, in their regulation. As PGC1α KO microarray data in this study show opposing findings to the PGC1α transgenic data, a loss-of-function experiment, as well as a gain-of-function experiment, revealed PGC1α's function in the oxidative energy metabolism of skeletal muscles. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a feather β-keratin gene exclusively expressed in pennaceous barbule cells of contour feathers in chicken.

PubMed

Kowata, Kinue; Nakaoka, Minori; Nishio, Kaori; Fukao, Ayaka; Satoh, Akira; Ogoshi, Maho; Takahashi, Sumio; Tsudzuki, Masaoki; Takeuchi, Sakae

2014-05-25

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of β-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BlSK1). The BlSK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BlSK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BlSK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken. Copyright © 2014 Elsevier B.V. All rights reserved.

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

PubMed

Xu, Yao; Zheng, Zhi

2016-05-15

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

[Alport syndrome: Hereditary nephropathy associated with mutations in genes coding for type IV collagen chains].

PubMed

Heidet, Laurence; Gubler, Marie-Claire

2016-12-01

Alport syndrome is an inherited disorder characterized by the association of a progressive haematuric nephropathy with ultrastructural abnormalities of the glomerular basement membranes, a progressive sensorineural hearing loss and sometimes ocular involvement. Its incidence is less than 1 per 5000 individuals and the disease is the cause of about 2% of end stage renal disease in Europe and the United States. Alport syndrome is clinically and genetically heterogeneous. It is related to mutations in the genes encoding one of three chains, α3, α4 α5 of type IV collagen, the main component of basement membranes, expressed in the glomerular basement membrane. COL4A5 mutations are associated with X-linked Alport syndrome, which represents 80 to 85% of cases and is more severe in boys than in girls. Mutations in COL4A3 or COL4A4 are associated with autosomal Alport syndrome. The expression of collagen chains in skin and kidney basement membranes allows for the diagnosis and characterization of the mode of transmission in most patients. It is necessary to diagnose this syndrome because its family involvement, its severity, and the importance of genetic counseling. Angiotensin blockers are increasingly prescribed in proteinuric patients. Prospective studies are needed to assess the effectiveness of these treatments on proteinuria and progression of kidney failure, and to specify indications. Animal studies have shown the potential value of different molecules (protease inhibitors, chemokine receptor blockers, transforming growth factor-β1 inhibitors, hydroxy-methyl-coenzyme A reductase inhibitors, bone morphogenetic protein-7 inhibitors), hematopoietic stem cells, and of a anti-micro-RNA. Copyright © 2016. Published by Elsevier SAS.

Novel genetic factors involved in resistance to Bacillus thuringiensis in Plutella xylostella.

PubMed

Ayra-Pardo, C; Raymond, B; Gulzar, A; Rodríguez-Cabrera, L; Morán-Bertot, I; Crickmore, N; Wright, D J

2015-12-01

The widespread and sustainable exploitation of the entomopathogen Bacillus thuringiensis (Bt) in pest control is threatened by the evolution of resistance. Although resistance is often associated with loss of binding of the Bt toxins to the insect midgut cells, other factors have been implicated. Here we used suppressive subtractive hybridization and gene expression suppression to identify additional molecular components involved in Bt-resistance in Plutella xylostella. We isolated transcripts from genes that were differentially expressed in the midgut of larvae from a resistant population, following ingestion of a Bt kurstaki HD1 strain-based commercial formulation (DiPel), and compared with a genetically similar susceptible population. Quantitative real-time polymerase-chain reaction (RT-PCR) analysis confirmed the differential basal expression of a subset of these genes. Gene expression suppression of three of these genes (P. xylostella cyclin-dependent kinase 5 regulatory subunit associated protein 1-like 1, stromal cell-derived factor 2-like 1 and hatching enzyme-like 1) significantly increased the pathogenicity of HD1 to the resistant population. In an attempt to link the multitude of factors reportedly influencing resistance to Bt with the well-characterized loss of toxin binding, we also considered Bt-resistance models in P. xylostella and other insects. © 2015 The Royal Entomological Society.

[Atopy and interleukin-4 receptor].

PubMed

Izuhara, K

1999-06-01

Both IL-4 and IL-13 induce IgE synthesis in B cells by binding to their functional receptors on target cells. These receptors are considered to be composed of heterodimers and both share the IL-4R alpha chain (IL-4R alpha) as a component. Atopy is an inherited tendency, underlying asthma, rhinitis and eczema, and generating high nonspecific IgE and/or high specific IgE against common antigens. Based on findings concerning the molecular mechanism of the signal transduction of IL-4 and IL-13, IL-4R alpha was considered a gene that gave rise to atopy. One polymorphism in the IL-4R alpha gene, Ile50Val, has been correlated with atopy by both genetic and functional assessment. The strategy used in these studies should lead to identification of other genes involved in atopy. Furthermore, these studies should be useful for gene diagnosis of atopy and development of new therapies for atopy in the future.

Molecular analysis of the ependymin gene and functional test of its promoter region by transient expression in Brachydanio rerio.

PubMed

Rinder, H; Bayer, T A; Gertzen, E M; Hoffmann, W

1992-01-01

Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.

Vitamin C modulates cadmium-induced hepatic antioxidants' gene transcripts and toxicopathic changes in Nile tilapia, Oreochromis niloticus.

PubMed

El-Sayed, Yasser S; El-Gazzar, Ahmed M; El-Nahas, Abeer F; Ashry, Khaled M

2016-01-01

Cadmium (Cd) is one of the naturally occurring heavy metals having adverse effects, while vitamin C (L-ascorbic acid) is an essential micronutrient for fish, which can attenuate tissue damage owing to its chain-breaking antioxidant and free radical scavenger properties. The adult Nile tilapia fish were exposed to Cd at 5 mg/l with and without vitamin C (500 mg/kg diet) for 45 days in addition to negative and positive controls fed with the basal diet and basal diet supplemented with vitamin C, respectively. Hepatic relative mRNA expression of genes involved in antioxidant function, metallothionein (MT), glutathione S-transferase (GST-α1), and glutathione peroxidase (GPx1), was assessed using real-time reverse transcription polymerase chain reaction (RT-PCR). Hepatic architecture was also histopathologically examined. Tilapia exposed to Cd exhibited upregulated antioxidants' gene transcript levels, GST-⍺1, GPx1, and MT by 6.10-, 4.60-, and 4.29-fold, respectively. Histopathologically, Cd caused severe hepatic changes of multifocal hepatocellular and pancreatic acinar necrosis, and lytic hepatocytes infiltrated with eosinophilic granular cells. Co-treatment of Cd-exposed fish with vitamin C overexpressed antioxidant enzyme-related genes, GST-⍺1 (16.26-fold) and GPx1 (18.68-fold), and maintained the expression of MT gene close to control (1.07-fold), averting the toxicopathic lesions induced by Cd. These results suggested that vitamin C has the potential to protect Nile tilapia from Cd hepatotoxicity via sustaining hepatic antioxidants' genes transcripts and normal histoarchitecture.

A Functional Approach towards Understanding the Role of the Mitochondrial Respiratory Chain in an Endomycorrhizal Symbiosis

PubMed Central

Mercy, Louis; Lucic-Mercy, Eva; Nogales, Amaia; Poghosyan, Areg; Schneider, Carolin; Arnholdt-Schmitt, Birgit

2017-01-01

Arbuscular mycorrhizal fungi (AMF) are crucial components of fertile soils, able to provide several ecosystem services for crop production. Current economic, social and legislative contexts should drive the so-called “second green revolution” by better exploiting these beneficial microorganisms. Many challenges still need to be overcome to better understand the mycorrhizal symbiosis, among which (i) the biotrophic nature of AMF, constraining their production, while (ii) phosphate acts as a limiting factor for the optimal mycorrhizal inoculum application and effectiveness. Organism fitness and adaptation to the changing environment can be driven by the modulation of mitochondrial respiratory chain, strongly connected to the phosphorus processing. Nevertheless, the role of the respiratory function in mycorrhiza remains largely unexplored. We hypothesized that the two mitochondrial respiratory chain components, alternative oxidase (AOX) and cytochrome oxidase (COX), are involved in specific mycorrhizal behavior. For this, a complex approach was developed. At the pre-symbiotic phase (axenic conditions), we studied phenotypic responses of Rhizoglomus irregulare spores with two AOX and COX inhibitors [respectively, salicylhydroxamic acid (SHAM) and potassium cyanide (KCN)] and two growth regulators (abscisic acid – ABA and gibberellic acid – Ga3). At the symbiotic phase, we analyzed phenotypic and transcriptomic (genes involved in respiration, transport, and fermentation) responses in Solanum tuberosum/Rhizoglomus irregulare biosystem (glasshouse conditions): we monitored the effects driven by ABA, and explored the modulations induced by SHAM and KCN under five phosphorus concentrations. KCN and SHAM inhibited in vitro spore germination while ABA and Ga3 induced differential spore germination and hyphal patterns. ABA promoted mycorrhizal colonization, strong arbuscule intensity and positive mycorrhizal growth dependency (MGD). In ABA treated plants, R. irregulare induced down-regulation of StAOX gene isoforms and up-regulation of genes involved in plant COX pathway. In all phosphorus (P) concentrations, blocking AOX or COX induced opposite mycorrhizal patterns in planta: KCN induced higher Arum-type arbuscule density, positive MGD but lower root colonization compared to SHAM, which favored Paris-type formation and negative MGD. Following our results and current state-of-the-art knowledge, we discuss metabolic functions linked to respiration that may occur within mycorrhizal behavior. We highlight potential connections between AOX pathways and fermentation, and we propose new research and mycorrhizal application perspectives. PMID:28424712

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

PubMed

Han, Rongchun; Takahashi, Hiroki; Nakamura, Michimi; Bunsupa, Somnuk; Yoshimoto, Naoko; Yamamoto, Hirobumi; Suzuki, Hideyuki; Shibata, Daisuke; Yamazaki, Mami; Saito, Kazuki

2015-01-01

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Application of HSVtk suicide gene to X-SCID gene therapy: Ganciclovir treatment offsets gene corrected X-SCID B cells

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

Uchiyama, Toru; Kumaki, Satoru; Ishikawa, Yoshinori

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human {gamma}c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the {gamma}c chain, the cells were treated with ganciclovir (GCV). The {gamma}c chain positive cells were eliminated under low concentrationmore » without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the {gamma}c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.« less

Identification of differentially expressed genes and signalling pathways in bark of Hevea brasiliensis seedlings associated with secondary laticifer differentiation using gene expression microarray.

PubMed

Loh, Swee Cheng; Thottathil, Gincy P; Othman, Ahmad Sofiman

2016-10-01

The natural rubber of Para rubber tree, Hevea brasiliensis, is the main crop involved in industrial rubber production due to its superior quality. The Hevea bark is commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. The laticifer is well defined in the aspect of morphology; however, only some genes associated with its development have been reported. We successfully induced secondary laticifer in the jasmonic acid (JA)-treated and linolenic acid (LA)-treated Hevea bark but secondary laticifer is not observed in the ethephon (ET)-treated and untreated Hevea bark. In this study, we analysed 27,195 gene models using NimbleGen microarrays based on the Hevea draft genome. 491 filtered differentially expressed (FDE) transcripts that are common to both JA- and LA-treated bark samples but not ET-treated bark samples were identified. In the Eukaryotic Orthologous Group (KOG) analysis, 491 FDE transcripts belong to different functional categories that reflect the diverse processes and pathways involved in laticifer differentiation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and KOG analysis, the profile of the FDE transcripts suggest that JA- and LA-treated bark samples have a sufficient molecular basis for secondary laticifer differentiation, especially regarding secondary metabolites metabolism. FDE genes in this category are from the cytochrome (CYP) P450 family, ATP-binding cassette (ABC) transporter family, short-chain dehydrogenase/reductase (SDR) family, or cinnamyl alcohol dehydrogenase (CAD) family. The data includes many genes involved in cell division, cell wall synthesis, and cell differentiation. The most abundant transcript in FDE list was SDR65C, reflecting its importance in laticifer differentiation. Using the Basic Local Alignment Search Tool (BLAST) as part of annotation and functional prediction, several characterised as well as uncharacterized transcription factors and genes were found in the dataset. Hence, the further characterization of these genes is necessary to unveil their role in laticifer differentiation. This study provides a platform for the further characterization and identification of the key genes involved in secondary laticifer differentiation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

PubMed

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

A tripartite clustering analysis on microRNA, gene and disease model.

PubMed

Shen, Chengcheng; Liu, Ying

2012-02-01

Alteration of gene expression in response to regulatory molecules or mutations could lead to different diseases. MicroRNAs (miRNAs) have been discovered to be involved in regulation of gene expression and a wide variety of diseases. In a tripartite biological network of human miRNAs, their predicted target genes and the diseases caused by altered expressions of these genes, valuable knowledge about the pathogenicity of miRNAs, involved genes and related disease classes can be revealed by co-clustering miRNAs, target genes and diseases simultaneously. Tripartite co-clustering can lead to more informative results than traditional co-clustering with only two kinds of members and pass the hidden relational information along the relation chain by considering multi-type members. Here we report a spectral co-clustering algorithm for k-partite graph to find clusters with heterogeneous members. We use the method to explore the potential relationships among miRNAs, genes and diseases. The clusters obtained from the algorithm have significantly higher density than randomly selected clusters, which means members in the same cluster are more likely to have common connections. Results also show that miRNAs in the same family based on the hairpin sequences tend to belong to the same cluster. We also validate the clustering results by checking the correlation of enriched gene functions and disease classes in the same cluster. Finally, widely studied miR-17-92 and its paralogs are analyzed as a case study to reveal that genes and diseases co-clustered with the miRNAs are in accordance with current research findings.

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy.

PubMed

Heo, Jin-Mi; Ordureau, Alban; Paulo, Joao A; Rinehart, Jesse; Harper, J Wade

2015-10-01

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

PubMed Central

Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

2016-01-01

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific ‘carbonic anhydrase domain’ of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe ‘life without complex I’. PMID:27122571

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

PubMed

Velázquez-López, Isabel; Valdés-García, Gilberto; Romero Romero, Sergio; Maya Martínez, Roberto; Leal-Cervantes, Ana I; Costas, Miguel; Sánchez-López, Rosana; Amero, Carlos; Pastor, Nina; Fernández Velasco, D Alejandro

2018-07-01

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

The Eurasian genes of the 2009 pandemic influenza virus: an integrative perspective on their conveyance to and assimilation in America.

PubMed

Shoham, Dany

2016-01-01

The formation of pandemic influenza genotypes varied phylogeographically and ecophylogenetically throughout their fully recognized recent 100-years natural history, involving consistently avian plus human genes, and at times swine genes. The last four traceable pandemic strains (PSs) included two American H1N1 viruses with genomes predominantly containing swine genes, of which at least one genome originated from both America and Eurasia; and two non-H1N1 Asian viruses with genomes entirely originating from Asia, and having no swine genes. This study explores whether there is a particular interhemispheric system underlying such divergence, and its properties. Unlike the assumption that transport of live pigs from Eurasia to America facilitated the formation of the 2009 H1N1 PS in America, it is suggested that conveyance of Eurasian swine genes to America, and their assimilation therein, took place through a distinct, perfectly natural ecophylogenetic machinery. The latter conjunctively involves, foremost, a native Asian duck-swine-man interface, a Holarctic chain of certain migratory Anas ducks, a native American turkey-swine-man interface, and two specific clades of American influenza A viruses. Likewise, the described machinery could have readily given rise to the 1918 H1N1, and, presumably, earlier American PSs, altogether constituting private cases of a much broader, self-sustained, permanent phylogeographic system.

Levels of potential bioactive compounds including carotenoids, vitamin C and phenolic compounds, and expression of their cognate biosynthetic genes vary significantly in different varieties of potato (Solanum tuberosum L.) grown under uniform cultural conditions.

PubMed

Valcarcel, Jesus; Reilly, Kim; Gaffney, Michael; O'Brien, Nora M

2016-02-01

In addition to their high carbohydrate content, potatoes are also an important dietary source of vitamin C and bioactive secondary metabolites, including phenolic compounds and carotenoids, which have been suggested to play a role in human health. The expression of genes encoding key enzymes involved in the synthesis of these compounds was assessed by reverse transcription-quantitative polymerase chain reaction and compared to the accumulation of the corresponding product in seven potato varieties showing contrasting levels of metabolite accumulation. Strong positive correlations were found between phenolic content in the flesh of tubers and transcript levels of phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. The expression of PAL and CHS was also related to that of AN1, a transcription factor involved in the synthesis of anthocyanins, suggesting that these genes are regulated in a coordinated manner. No clear relationship was found between transcript levels of phytoene synthase (PSY) or L-galactono-1,4-lactone dehydrogenase (GLDH) genes and total carotenoid or vitamin C accumulation, respectively. Data indicate that levels of total phenolic and flavonoid compounds in potato are controlled primarily by PAL and CHS gene expression. Transcript levels of PSY and GLDH did not control accumulation of carotenoids or vitamin C. © 2015 Society of Chemical Industry.

Identification, Characterization and Expression of Methuselah-Like Genes in Dastarcus helophoroides (Coleoptera: Bothrideridae).

PubMed

Zhang, Zhengqing; Wang, Huapeng; Hao, Chunfeng; Zhang, Wei; Yang, Miaomiao; Chang, Yong; Li, Menglou

2016-10-21

Dastarcus helophoroides , which has a relatively longer lifespan compared to other insects, is one of the most effective natural enemies of many large-body long-horned beetles. Methuselah (Mth) is associated with the lifespan, stress resistance, and reproduction in Drosophila melanogaster , but Mth is not present in non-drosophiline insects. A number of methuselah-like genes ( mth-likes , mthls ) have been identified in non-drosophiline insects, but it is still unknown whether they are present in Dastarcus helophoroides . We identified three novel mth-like genes in D. helophoroides : mth-like1 , mth-like2 , and mth-like5 , and carried out bioinformatic analysis based on the full-length nucleic acid sequences and deduced amino acid sequences. Real-time quantitative polymerase chain reaction (RT-qPCR) showed variations in expression patterns of mth-like genes in different tissues (highly expressed in reproductive systems) and at different developmental stages, indicating that mth-likes were likely be involved in reproduction and development. The altered mRNA expression in aging adults and under oxidation, high temperature, and starvation stress, indicated that mth-like genes were likely to be involved in aging and the resistance of oxidation, high temperature, and starvation. These results characterize, for the first time, the basic properties of three mth-like genes from D. helophoroides that probably play important roles in development, aging, reproduction, and stress resistance.

Insights into the Microbial Degradation of Rubber and Gutta-Percha by Analysis of the Complete Genome of Nocardia nova SH22a

PubMed Central

Luo, Quan; Hiessl, Sebastian; Poehlein, Anja; Daniel, Rolf

2014-01-01

The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid–AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity. PMID:24747905

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress.

PubMed

Dutilleul, Christelle; Chavarria, Heidy; Rézé, Nathalie; Sotta, Bruno; Baudouin, Emmanuel; Guillas, Isabelle

2015-12-01

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress. © 2015 John Wiley & Sons Ltd.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference.

PubMed

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference

PubMed Central

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path. PMID:26544851

[Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro].

PubMed

Yurova, K A; Sokhonevich, N A; Khaziakhmatova, O G; Litvinova, L S

2016-01-01

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

PubMed

Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

1998-08-28

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Beta-lactamic resistance profiles in Porphyromonas, Prevotella, and Parvimonas species isolated from acute endodontic infections.

PubMed

Montagner, Francisco; Jacinto, Rogério Castilho; Correa Signoretti, Fernanda Graziela; Scheffer de Mattos, Vanessa; Grecca, Fabiana Soares; Gomes, Brenda Paula Figueiredo de Almeida

2014-03-01

Susceptibility to beta-lactamic agents has changed among anaerobic isolates from acute endodontic infections. The aim of the present study was to determine the prevalence of the cfxA/cfxA2 gene in Prevotella spp., Porphyromonas spp., and Parviomonas micra strains and show its phenotypic expression. Root canal samples from teeth with acute endodontic infections were collected and Porphyromonas, Prevotella, and Parvimonas micra strains were isolated and microbiologically identified with conventional culture techniques. The susceptibility of the isolates was determined by the minimum inhibitory concentration of benzylpenicillin, amoxicillin, and amoxicillin + clavulanate using the E-test method (AB BIODISK, Solna, Sweden). The presence of the cfxA/cfxA2 gene was determined through primer-specific polymerase chain reaction. The nitrocefin test was used to determine the expression of the lactamase enzyme. Prevotella disiens, Prevotella oralis, Porphyromonas gingivalis, and P. micra strains were susceptible to benzylpenicillin, amoxicillin, and amoxicillin + clavulanate. The cfxA/cfxA2 gene was detected in 2 of 29 isolates (6.9%). Simultaneous detection of the cfxA/cfxA2 gene and lactamase production was observed for 1 Prevotella buccalis strain. The gene was in 1 P. micra strain but was not expressed. Three strains were positive for lactamase production, but the cfxA/cfxA2 gene was not detected through polymerase chain reaction. There is a low prevalence of the cfxA/cfxA2 gene and its expression in Porphyromonas spp., Prevotella spp., and P. micra strains isolated from acute endodontic infections. Genetic and phenotypic screening must be performed simultaneously to best describe additional mechanisms involved in lactamic resistance for strict anaerobes. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

PubMed Central

Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

2012-01-01

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

Gene duplication and the evolution of hemoglobin isoform differentiation in birds.

PubMed

Grispo, Michael T; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Storz, Jay F

2012-11-02

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.

Insights into the role of differential gene expression on the ecological adaptation of the snail Littorina saxatilis

PubMed Central

2010-01-01

Background In the past 40 years, there has been increasing acceptance that variation in levels of gene expression represents a major source of evolutionary novelty. Gene expression divergence is therefore likely to be involved in the emergence of incipient species, namely, in a context of adaptive radiation. In this study, a genome-wide expression profiling approach (cDNA-AFLP), validated by quantitative real-time polymerase chain reaction (qPCR) were used to get insights into the role of differential gene expression on the ecological adaptation of the marine snail Littorina saxatilis. This gastropod displays two sympatric ecotypes (RB and SU) which are becoming one of the best studied systems for ecological speciation. Results Among the 99 transcripts shared between ecotypes, 12.12% showed significant differential expression. At least 4% of these transcripts still displayed significant differences after correction for multiple tests, highlighting that gene expression can differ considerably between subpopulations adapted to alternative habitats in the face of gene flow. One of the transcripts identified was Cytochrome c Oxidase subunit I (COI). In addition, 6 possible reference genes were validated to normalize and confirm this result using qPCR. α-Tubulin and histone H3.3 showed the more stable expression levels, being therefore chosen as the best option for normalization. The qPCR analysis confirmed a higher COI expression in SU individuals. Conclusions At least 4% of the transcriptome studied is being differentially expressed between ecotypes living in alternative habitats, even when gene flow is still substantial between ecotypes. We could identify a candidate transcript of such ecotype differentiation: Cytochrome c Oxidase Subunit I (COI), a mitochondrial gene involved in energy metabolism. Quantitative PCR was used to confirm the differences found in COI and its over-expression in the SU ecotype. Interestingly, COI is involved in the oxidative phosphorylation, suggesting an enhanced mitochondrial gene expression (or increased number of mitochondria) to improve energy supply in the ecotype subjected to the strongest wave action. PMID:21087461

PaeX, a Second Pectin Acetylesterase of Erwinia chrysanthemi 3937

PubMed Central

Shevchik, Vladimir E.; Hugouvieux-Cotte-Pattat, Nicole

2003-01-01

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin. PMID:12730169

PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937.

PubMed

Shevchik, Vladimir E; Hugouvieux-Cotte-Pattat, Nicole

2003-05-01

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

Polymerase Chain Reaction (PCR)-based methods for detection and identification of mycotoxigenic Penicillium species using conserved genes

USDA-ARS?s Scientific Manuscript database

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of d...

Genes associated with thermosensitive genic male sterility in rice identified by comparative expression profiling.

PubMed

Pan, Yufang; Li, Qiaofeng; Wang, Zhizheng; Wang, Yang; Ma, Rui; Zhu, Lili; He, Guangcun; Chen, Rongzhi

2014-12-16

Thermosensitive genic male sterile (TGMS) lines and photoperiod-sensitive genic male sterile (PGMS) lines have been successfully used in hybridization to improve rice yields. However, the molecular mechanisms underlying male sterility transitions in most PGMS/TGMS rice lines are unclear. In the recently developed TGMS-Co27 line, the male sterility is based on co-suppression of a UDP-glucose pyrophosphorylase gene (Ugp1), but further study is needed to fully elucidate the molecular mechanisms involved. Microarray-based transcriptome profiling of TGMS-Co27 and wild-type Hejiang 19 (H1493) plants grown at high and low temperatures revealed that 15462 probe sets representing 8303 genes were differentially expressed in the two lines, under the two conditions, or both. Environmental factors strongly affected global gene expression. Some genes important for pollen development were strongly repressed in TGMS-Co27 at high temperature. More significantly, series-cluster analysis of differentially expressed genes (DEGs) between TGMS-Co27 plants grown under the two conditions showed that low temperature induced the expression of a gene cluster. This cluster was found to be essential for sterility transition. It includes many meiosis stage-related genes that are probably important for thermosensitive male sterility in TGMS-Co27, inter alia: Arg/Ser-rich domain (RS)-containing zinc finger proteins, polypyrimidine tract-binding proteins (PTBs), DEAD/DEAH box RNA helicases, ZOS (C2H2 zinc finger proteins of Oryza sativa), at least one polyadenylate-binding protein and some other RNA recognition motif (RRM) domain-containing proteins involved in post-transcriptional processes, eukaryotic initiation factor 5B (eIF5B), ribosomal proteins (L37, L1p/L10e, L27 and L24), aminoacyl-tRNA synthetases (ARSs), eukaryotic elongation factor Tu (eEF-Tu) and a peptide chain release factor protein involved in translation. The differential expression of 12 DEGs that are important for pollen development, low temperature responses or TGMS was validated by quantitative RT-PCR (qRT-PCR). Temperature strongly affects global gene expression and may be the common regulator of fertility in PGMS/TGMS rice lines. The identified expression changes reflect perturbations in the transcriptomic regulation of pollen development networks in TGMS-Co27. Findings from this and previous studies indicate that sets of genes involved in post-transcriptional and translation processes are involved in thermosensitive male sterility transitions in TGMS-Co27.

Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction

PubMed Central

Pedrini, Nicolás; Ortiz-Urquiza, Almudena; Huarte-Bonnet, Carla; Zhang, Shizhu; Keyhani, Nemat O.

2013-01-01

Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and four CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of six genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon synthesis. PMID:23422735

Genome-Wide Analysis of C2H2 Zinc-Finger Family Transcription Factors and Their Responses to Abiotic Stresses in Poplar (Populus trichocarpa)

PubMed Central

Liu, Quangang; Wang, Zhanchao; Xu, Xuemei; Zhang, Haizhen; Li, Chenghao

2015-01-01

Background C2H2 zinc-finger (C2H2-ZF) proteins are a large gene family in plants that participate in various aspects of normal plant growth and development, as well as in biotic and abiotic stress responses. To date, no overall analysis incorporating evolutionary history and expression profiling of the C2H2-ZF gene family in model tree species poplar (Populus trichocarpa) has been reported. Principal Findings Here, we identified 109 full-length C2H2-ZF genes in P. trichocarpa, and classified them into four groups, based on phylogenetic analysis. The 109 C2H2-ZF genes were distributed unequally on 19 P. trichocarpa linkage groups (LGs), with 39 segmental duplication events, indicating that segmental duplication has been important in the expansion of the C2H2-ZF gene family. Promoter cis-element analysis indicated that most of the C2H2-ZF genes contain phytohormone or abiotic stress-related cis-elements. The expression patterns of C2H2-ZF genes, based on heatmap analysis, suggested that C2H2-ZF genes are involved in tissue and organ development, especially root and floral development. Expression analysis based on quantitative real-time reverse transcription polymerase chain reaction indicated that C2H2-ZF genes are significantly involved in drought, heat and salt response, possibly via different mechanisms. Conclusions This study provides a thorough overview of the P. trichocarpa C2H2-ZF gene family and presents a new perspective on the evolution of this gene family. In particular, some C2H2-ZF genes may be involved in environmental stress tolerance regulation. PtrZFP2, 19 and 95 showed high expression levels in leaves and/or roots under environmental stresses. Additionally, this study provided a solid foundation for studying the biological roles of C2H2-ZF genes in Populus growth and development. These results form the basis for further investigation of the roles of these candidate genes and for future genetic engineering and gene functional studies in Populus. PMID:26237514

[Application of the polymerase chain reaction (PCR) in the diagnosis of Hb S-beta(+)-thalassemia].

PubMed

Harano, K; Harano, T; Kushida, Y; Ueda, S

1991-08-01

Isoelectric focusing of the hemolysate prepared from a two-year-old American black boy with microcytic hypochromia showed the presence of a high percentage (63.3%) of such Hb variant as Hb S, while the levels of Hb A, Hb F and Hb A2 were 20.0%, 12.7%, and 4.0%, respectively. The ratio of the non-alpha-chain to the alpha-chain of the biosynthesized globin chains was 0.49. The variant was identified as Hb S by amino acid analysis of the abnormal peptide (beta T-1) and digestion of DNA amplified by the polymerase chain reaction with enzyme Eco 81 I. This was further confirmed by DNA sequencing. DNA sequencing of a beta-gene without the beta s-mutation revealed a nucleotide change of T to C in the polyadenylation signal sequence AATAAA 3' to the beta-gene, resulting in beta(+)-thalassemia. These results are consistent with the existence of a beta s-gene and a beta(+)-thalassemia gene in trans.

Up-regulation of the G3PDH 'housekeeping' gene by estrogen.

PubMed

Galal, Nadia; El-Beialy, Waleed; Deyama, Yoshiaki; Yoshimura, Yoshitaka; Tei, Kanchu; Suzuki, Kuniaki; Totsuka, Yasunori

2010-01-01

Proteomic and genomic studies commonly involve the assessment of mRNA levels using reverse transcription-polymerase chain reaction (PCR) and real-time quantitative PCR. An internal standard RNA is fundamentally analyzed along with the investigated mRNA to document the specificity of the effect(s) on mRNA and to correct for inter-sample variations. In our studies implementing estrogen treatments on different cell lines, we initially used glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal standard. However, the results of PCR amplification demonstrated that 17β-estradiol enhanced the expression of the G3PDH gene, rendering it impossible to use G3PDH as an unbiased comparative control.

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

PubMed Central

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects. PMID:27547033

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells.

PubMed

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

Copy Number Variation of TLR-7 Gene and its Association with the Development of Systemic Lupus Erythematosus in Female Patients from Yucatan Mexico

PubMed Central

Pacheco, Guillermo Valencia; Cruz, Darig Cámara; González Herrera, Lizbeth J; Pérez Mendoza, Gerardo J; Adrián Amaro, Guadalupe I; Nakazawa Ueji, Yumi E; Angulo Ramírez, Angélica V

2014-01-01

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies against self-antigens, which occurs most often in women between 15 and 40 years of age. The innate immunity is involved in the pathogenesis of SLE through TLR- 7. Genetic factors such as copy number variation (CNV) of target genes may contribute to disease development, but this possible risk has not yet been studied in SLE patients from Yucatan, Mexico. The CNV of TLR-7 gene was determined by quantitative polymerase chain reaction assay using TaqMan probes in 80 SLE women and 150 control subjects. The results showed that 10% of SLE patients exhibited more than two copies of TLR-7 gene, whereas no mRNA overexpression was detected. These data suggested that increased CNV of the TLR-7 gene in Yucatan SLE women can be a risk factor for this disease. PMID:25512712

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress

PubMed Central

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-01-01

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance. PMID:28417911

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress.

PubMed

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-04-12

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance.

Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro-aerobic conditions.

PubMed

Liu, Jingjing; Sun, Faqian; Wang, Liang; Ju, Xi; Wu, Weixiang; Chen, Yingxu

2014-01-01

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro-aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real-time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro-aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation*

PubMed Central

Wakashima, Takeshi; Abe, Kensuke; Kihara, Akio

2014-01-01

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P. PMID:25049234

Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

PubMed

Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

2018-05-05

BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows.

PubMed

Sadri, H; Bruckmaier, R M; Rahmani, H R; Ghorbani, G R; Morel, I; van Dorland, H A

2010-10-01

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFα), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, β-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFα concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement. © 2010 Blackwell Verlag GmbH.

Form-Deprivation Myopia in Chick Induces Limited Changes in Retinal Gene Expression

PubMed Central

McGlinn, Alice M.; Baldwin, Donald A.; Tobias, John W.; Budak, Murat T.; Khurana, Tejvir S.; Stone, Richard A.

2007-01-01

Purpose Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick. Methods One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription–polymerase chain reaction (qPCR) in independent biological replicates. Results Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II–related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated. Conclusions Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development. PMID:17652709

Vaginal Gene Expression During Treatment With Aromatase Inhibitors.

PubMed

Kallak, Theodora Kunovac; Baumgart, Juliane; Nilsson, Kerstin; Åkerud, Helena; Poromaa, Inger Sundström; Stavreus-Evers, Anneli

2015-12-01

Aromatase inhibitor (AI) treatment suppresses estrogen biosynthesis and causes genitourinary symptoms of menopause such as vaginal symptoms, ultimately affecting the quality of life for many postmenopausal women with breast cancer. Thus, the aim of this study was to examine vaginal gene expression in women during treatment with AIs compared with estrogen-treated women. The secondary aim was to study the presence and localization of vaginal aromatase. Vaginal biopsies were collected from postmenopausal women treated with AIs and from age-matched control women treated with vaginal estrogen therapy. Differential gene expression was studied with the Affymetrix Gene Chip Gene 1.0 ST Array (Affymetrix Inc, Santa Clara, CA) system, Ingenuity pathway analysis, quantitative real-time polymerase chain reaction, and immunohistochemistry. The expression of 279 genes differed between the 2 groups; AI-treated women had low expression of genes involved in cell differentiation, proliferation, and cell adhesion. Some differentially expressed genes were found to interact indirectly with the estrogen receptor alpha. In addition, aromatase protein staining was evident in the basal and the intermediate vaginal epithelium layers, and also in stromal cells with a slightly stronger staining intensity found in AI-treated women. In this study, we demonstrated that genes involved in cell differentiation, proliferation, and cell adhesion are differentially expressed in AI-treated women. The expression of vaginal aromatase suggests that this could be the result of local and systemic inhibition of aromatase. Our results emphasize the role of estrogen for vaginal cell differentiation and proliferation and future drug candidates should be aimed at improving cell differentiation and proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

Phylogeny of Rieske/cytb Complexes with a Special Focus on the Haloarchaeal Enzymes

PubMed Central

Baymann, Frauke; Schoepp-Cothenet, Barbara; Lebrun, Evelyne; van Lis, Robert; Nitschke, Wolfgang

2012-01-01

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable. PMID:22798450

Poultry hatcheries as potential reservoirs for antimicrobial-resistant Escherichia coli: A risk to public health and food safety.

PubMed

Osman, Kamelia M; Kappell, Anthony D; Elhadidy, Mohamed; ElMougy, Fatma; El-Ghany, Wafaa A Abd; Orabi, Ahmed; Mubarak, Aymen S; Dawoud, Turki M; Hemeg, Hassan A; Moussa, Ihab M I; Hessain, Ashgan M; Yousef, Hend M Y

2018-04-11

Hatcheries have the power to spread antimicrobial resistant (AMR) pathogens through the poultry value chain because of their central position in the poultry production chain. Currently, no information is available about the presence of AMR Escherichia coli strains and the antibiotic resistance genes (ARGs) they harbor within hatchezries. Therefore, this study aimed to investigate the possible involvement of hatcheries in harboring hemolytic AMR E. coli. Serotyping of the 65 isolated hemolytic E. coli revealed 15 serotypes with the ability to produce moderate biofilms, and shared susceptibility to cephradine and fosfomycin and resistance to spectinomycin. The most common β-lactam resistance gene was bla TEM , followed by bla OXA-1 , bla MOX -like , bla CIT -like , bla SHV and bla FOX . Hierarchical clustering of E. coli isolates based on their phenotypic and genotypic profiles revealed separation of the majority of isolates from hatchlings and the hatchery environments, suggesting that hatchling and environmental isolates may have different origins. The high frequency of β-lactam resistance genes in AMR E. coli from chick hatchlings indicates that hatcheries may be a reservoir of AMR E. coli and can be a major contributor to the increased environmental burden of ARGs posing an eminent threat to poultry and human health.

Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses.

PubMed

Heo, W D; Lee, S H; Kim, M C; Kim, J C; Chung, W S; Chun, H J; Lee, K J; Park, C Y; Park, H C; Choi, J Y; Cho, M J

1999-01-19

The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Novel mutations in the genes TGM1 and ALOXE3 underlying autosomal recessive congenital ichthyosis

PubMed Central

Ullah, Rahim; Ansar, Muhammad; Durrani, Zaka Ullah; Lee, Kwanghyuk; Santos-Cortez, Regie Lyn P.; Muhammad, Dost; Ali, Mahboob; Zia, Muhammad; Ayub, Muhammad; Khan, Suliman; Smith, Josh D.; Nickerson, Deborah A.; Shendure, Jay; Bamshad, Michael; Leal, Suzanne M.; Ahmad, Wasim

2016-01-01

Background Ichthyoses are clinically characterized by scaling or hyperkeratosis of the skin or both. It can be an isolated condition limited to the skin or appear secondarily with involvement of other cutaneous or systemic abnormalities. Methods The present study investigated clinical and molecular characterization of three consanguineous families (A, B, C) segregating two different forms of autosomal recessive congenital ichthyosis (ARCI). Linkage in three consanguineous families (A, B, C) segregating two different forms of ARCI was searched by typing microsatellite and single nucleotide polymorphism marker analysis. Sequencing of the two genes TGM1 and ALOXE3 was performed by the dideoxy chain termination method. Results Genome-wide linkage analysis established linkage in family A to TGM1 gene on chromosome 14q11 and in families B and C to ALOXE3 gene on chromosome 17p13. Subsequently, sequencing of these genes using samples from affected family members led to the identification of three novel mutations: a missense variant p.Trp455Arg in TGM1 (family A); a nonsense variant p.Arg140* in ALOXE3 (family B); and a complex rearrangement in ALOXE3 (family C). Conclusion The present study further extends the spectrum of mutations in the two genes involved in causing ARCI. Characterizing the clinical spectrum resulting from mutations in the TGM1 and ALOXE3 genes will improve diagnosis and may direct clinical care of the family members. PMID:26578203

Conservation of globin genes in the "living fossil" Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family.

PubMed

Schwarze, Kim; Burmester, Thorsten

2013-09-01

The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the "living fossil" Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a "globin fossil". Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains - with the exception of a more complex pattern in the coelacanth and amphibians - display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of globins in certain taxa may be associated with changes in the oxygen-dependent metabolism. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Shark Ig light chain junctions are as diverse as in heavy chains.

PubMed

Fleurant, Marshall; Changchien, Lily; Chen, Chin-Tung; Flajnik, Martin F; Hsu, Ellen

2004-11-01

We have characterized a small family of four genes encoding one of the three nurse shark Ig L chain isotypes, called NS5. All NS5 cDNA sequences are encoded by three loci, of which two are organized as conventional clusters, each consisting of a V and J gene segment that can recombine and one C region exon; the third contains a germline-joined VJ in-frame and the fourth locus is a pseudogene. This is the second nurse shark L chain type where both germline-joined and split V-J organizations have been found. Since there are only two rearranging Ig loci, it was possible for the first time to examine junctional diversity in defined fish Ig genes, comparing productive vs nonproductive rearrangements. N region addition was found to be considerably more extensive in length and in frequency than any other vertebrate L chain so far reported and rivals that in H chain. We put forth the speculation that the unprecedented efficiency of N region addition (87-93% of NS5 sequences) may be a result not only of simultaneous H and L chain rearrangement in the shark but also of processing events that afford greater accessibility of the V or J gene coding ends to terminal deoxynucleotidyltransferase.

MHC II-β chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

PubMed

Picchietti, S; Abelli, L; Guerra, L; Randelli, E; Proietti Serafini, F; Belardinelli, M C; Buonocore, F; Bernini, C; Fausto, A M; Scapigliati, G

2015-02-01

MHC II-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sequence homology and expression profile of genes associated with DNA repair pathways in Mycobacterium leprae.

PubMed

Sharma, Mukul; Vedithi, Sundeep Chaitanya; Das, Madhusmita; Roy, Anindya; Ebenezer, Mannam

2017-01-01

Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae. T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%), 11 hypothetical proteins (18%), and 14 pseudogenes (23%). All these genes have homologs in M. tuberculosis and 49 (80.32%) in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps. It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA). The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes) were analyzed using quantitative Polymerase Chain Reaction (qPCR) assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the direct repair pathway. This study provided preliminary information on the potential DNA repair pathways that are extant in M. leprae and the associated genes.

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

PubMed

Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

2016-08-01

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

PubMed Central

Calvo, Sarah E; Tucker, Elena J; Compton, Alison G; Kirby, Denise M; Crawford, Gabriel; Burtt, Noel P; Rivas, Manuel A; Guiducci, Candace; Bruno, Damien L; Goldberger, Olga A; Redman, Michelle C; Wiltshire, Esko; Wilson, Callum J; Altshuler, David; Gabriel, Stacey B; Daly, Mark J; Thorburn, David R; Mootha, Vamsi K

2010-01-01

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing, and experimental validation to uncover the molecular basis of mitochondrial complex I (CI) disorders. We created five pools of DNA from a cohort of 103 patients and then performed deep sequencing of 103 candidate genes to spotlight 151 rare variants predicted to impact protein function. We used confirmatory experiments to establish genetic diagnoses in 22% of previously unsolved cases, and discovered that defects in NUBPL and FOXRED1 can cause CI deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can reveal novel disease-causing mutations in individual patients. PMID:20818383

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Transcription profile data of phorbol esters biosynthetic genes during developmental stages in Jatropha curcas.

PubMed

Jadid, Nurul; Mardika, Rizal Kharisma; Purwani, Kristanti Indah; Permatasari, Erlyta Vivi; Prasetyowati, Indah; Irawan, Mohammad Isa

2018-06-01

Jatropha curcas is currently known as an alternative source for biodiesel production. Beside its high free fatty acid content, J. curcas also contains typical diterpenoid-toxic compounds of Euphorbiaceae plant namely phorbol esters. This article present the transcription profile data of genes involved in the biosynthesis of phorbol esters at different developmental stages of leaves, fruit, and seed in Jatropha curcas . Transcriptional profiles were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). We used two genes including GGPPS (Geranylgeranyl diphospate synthase), which is responsible for the formation of common diterpenoid precursor (GGPP) and CS (Casbene Synthase), which functions in the synthesis of casbene. Meanwhile, J. curcas Actin ( ACT ) was used as internal standard. We demonstrated dynamic of GGPPS and CS expression among different stage of development of leaves, fruit and seed in Jatropha .

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6▿ †

PubMed Central

Sato, Yusuke; Moriuchi, Hideki; Hishiyama, Shojiro; Otsuka, Yuichiro; Oshima, Kenji; Kasai, Daisuke; Nakamura, Masaya; Ohara, Seiji; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

2009-01-01

Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (αR,βS)-GGE and (αR,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (αS,βR)-GGE and (αS,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (αR,βS)-GGE and (αR,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers. PMID:19542348

Structure and dynamics of solvated polyethylenimine chains

NASA Astrophysics Data System (ADS)

Beu, Titus A.; Farcaş, Alexandra

2017-12-01

Polimeric gene-delivery carriers have attracted great interest in recent years, owing to their applicability in gene therapy. In particular, cationic polymers represent the most promising delivery vectors for nucleic acids into the cells. This study presents extensive atomistic molecular dynamics simulations of linear polyethylenimine chains. The simulations show that the variation of the chain size and protonation fraction causes a substantial change of the diffusion coefficient. Examination of the solvated chains suggests the possibility of controlling the polymer diffusion mobility in solution.

Identifying pathogenic processes by integrating microarray data with prior knowledge

PubMed Central

2014-01-01

Background It is of great importance to identify molecular processes and pathways that are involved in disease etiology. Although there has been an extensive use of various high-throughput methods for this task, pathogenic pathways are still not completely understood. Often the set of genes or proteins identified as altered in genome-wide screens show a poor overlap with canonical disease pathways. These findings are difficult to interpret, yet crucial in order to improve the understanding of the molecular processes underlying the disease progression. We present a novel method for identifying groups of connected molecules from a set of differentially expressed genes. These groups represent functional modules sharing common cellular function and involve signaling and regulatory events. Specifically, our method makes use of Bayesian statistics to identify groups of co-regulated genes based on the microarray data, where external information about molecular interactions and connections are used as priors in the group assignments. Markov chain Monte Carlo sampling is used to search for the most reliable grouping. Results Simulation results showed that the method improved the ability of identifying correct groups compared to traditional clustering, especially for small sample sizes. Applied to a microarray heart failure dataset the method found one large cluster with several genes important for the structure of the extracellular matrix and a smaller group with many genes involved in carbohydrate metabolism. The method was also applied to a microarray dataset on melanoma cancer patients with or without metastasis, where the main cluster was dominated by genes related to keratinocyte differentiation. Conclusion Our method found clusters overlapping with known pathogenic processes, but also pointed to new connections extending beyond the classical pathways. PMID:24758699

Transcriptome Profiling of Radish (Raphanus sativus L.) Root and Identification of Genes Involved in Response to Lead (Pb) Stress with Next Generation Sequencing

PubMed Central

Wang, Yan; Xu, Liang; Chen, Yinglong; Shen, Hong; Gong, Yiqin; Limera, Cecilia; Liu, Liwang

2013-01-01

Lead (Pb), one of the most toxic heavy metals, can be absorbed and accumulated by plant roots and then enter the food chain resulting in potential health risks for human beings. The radish (Raphanus sativus L.) is an important root vegetable crop with fleshy taproots as the edible parts. Little is known about the mechanism by which radishes respond to Pb stress at the molecular level. In this study, Next Generation Sequencing (NGS)–based RNA-seq technology was employed to characterize the de novo transcriptome of radish roots and identify differentially expressed genes (DEGs) during Pb stress. A total of 68,940 assembled unique transcripts including 33,337 unigenes were obtained from radish root cDNA samples. Based on the assembled de novo transcriptome, 4,614 DEGs were detected between the two libraries of untreated (CK) and Pb-treated (Pb1000) roots. Gene Ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Pb stress are predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to heavy metal Pb. Identification of potential DEGs involved in responses to Pb stress significantly reflected alterations in major biological processes and metabolic pathways. The molecular basis of the response to Pb stress in radishes was comprehensively characterized. Useful information and new insights were provided for investigating the molecular regulation mechanism of heavy metal Pb accumulation and tolerance in root vegetable crops. PMID:23840502

Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

PubMed

Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

2015-04-01

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

Detection of Clostridium botulinum type C cells in the gastrointestinal tracts of Mozambique tilapia (Oreochromis mossambicus) by polymerase chain reaction

USGS Publications Warehouse

Nol, P.; Williamson, J.L.; Rocke, T.E.; Yuill, Thomas M.

2004-01-01

We established a method of directly detecting Clostridium botulinum type C cells, while minimizing spore detection, in the intestinal contents of Mozambique tilapia (Oreochromis mossambicus). This technique involved extraction of predominantly cellular DNA from tilapia intestinal tracts and used a polymerase chain reaction assay to detect presence of type C1 toxin gene. We consistently detected C. botulinum type C cells in tilapia gastrointestinal contents at a level of 7.5×104 cells per 0.25 g material or 1.9×103 cells. This technique is useful for determining prevalence of the potentially active organisms within a given population of fish and may be adapted to other types of C. botulinum and vertebrate populations as well.

Clonal T-Cell Receptor γ-Chain Gene Rearrangements in Differential Diagnosis of Lymphomatoid Papulosis From Skin Metastasis of Nodal Anaplastic Large-Cell Lymphoma

PubMed Central

Akilov, Oleg E.; Pillai, Raju K.; Grandinetti, Lisa M.; Kant, Jeffrey A.; Geskin, Larisa

2012-01-01

Background In patients with a history of nodal anaplastic large-cell lymphoma (ALCL), differentiation of type C lymphomatoid papulosis from cutaneous involvement of systemic ALCL may be challenging because the 2 entities may exhibit identical histologic features. Although metastatic ALCL generally carries the same clone as the primary lymphoma, expression of a distinct clone likely represents a distinct process. Observations A 54-year-old white man had a history of anaplastic lymphoma kinase 1–negative ALCL in the right inguinal lymph node 6 years ago. A complete response was achieved after 6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine [Oncovin], and prednisone administered in 21-day cycles) and radiation therapy. After 3½ years, the patient observed waxing and waning papules and nodules. Examination of the biopsy specimen revealed a dense CD30+ lymphocytic infiltrate; no evidence of systemic malignancy was evident on positron emission tomography. Although clinically the presentation was consistent with lymphomatoid papulosis, metastatic ALCL had to be excluded. Polymerase chain reaction analysis with T-cell receptor γ-chain gene rearrangement (TCR-γR) was performed on the original lymph node and new skin lesions. Results of the TCR-γR analysis were positive for clonality in both lesions. However, separate clonal processes were identified. The identification of distinct clones supported the clinical impression of lymphomatoid papulosis. Conclusion Polymerase chain reaction analysis of TCR-γR is a useful method for distinguishing different clonal processes and is recommended when differentiation of primary and secondary lymphoproliferative disorders is required. PMID:21844453

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus (MRSA) along the production chain of dairy products in north-western Greece.

PubMed

Papadopoulos, Panagiotis; Papadopoulos, Theofilos; Angelidis, Apostolos S; Boukouvala, Evridiki; Zdragas, Antonios; Papa, Anna; Hadjichristodoulou, Christos; Sergelidis, Daniel

2018-02-01

The objective of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the production chain of dairy products. Of 367 tested samples (36 bulk tank milk (BTM), 19 dairy products, 72 human, 185 animal, 55 equipment), 212 (57.8%) were found positive for S. aureus. Almost all isolates (99.6%) were resistant to at least one antimicrobial and 13.3% were multi-drug resistant (MDR), exhibiting resistance to three or more antibiotic classes. Eleven samples (3%) were found contaminated by MRSA carrying the mecA gene. None of the MRSA isolates carried the mecC or the Pandon-Valentine leucocidin (PVL) genes. Four spa types were identified among the MRSA isolates: t127, t3586, t1773, t4038, with t127 being the most prevalent (7 out of 11). Two of them, t3586 and t1773, were isolated for the first time in Greece. Furthermore, Pulse-Field Gel Electrophoresis (PFGE) analysis indicated clonal circulation through the dairy production chain. The presence of MDR S. aureus, and especially MRSA, in animals and dairy products represents a potential threat for the spread of this pathogen in the community. The results indicated that human, animal and environmental sources could be involved in the contamination of dairy products along their production chain and therefore further investigation of contamination sources is needed to control the dispersion of MRSA in the community. Copyright © 2017 Elsevier Ltd. All rights reserved.

Copy Number Variations of TBK1 in Australian Patients With Primary Open-Angle Glaucoma

PubMed Central

AWADALLA, MONA S.; FINGERT, JOHN H.; ROOS, BENJAMIN E.; CHEN, SIMON; HOLMES, RICHARD; GRAHAM, STUART L.; CHEHADE, MARK; GALANOPOLOUS, ANNA; RIDGE, BRONWYN; SOUZEAU, EMMANUELLE; ZHOU, TIGER; SIGGS, OWEN M.; HEWITT, ALEX W.; MACKEY, DAVID A.; BURDON, KATHRYN P.; CRAIG, JAMIE E.

2015-01-01

PURPOSE To investigate the presence of TBK1 copy number variations in a large, well-characterized Australian cohort of patients with glaucoma comprising both normal-tension glaucoma and high-tension glaucoma cases. DESIGN A retrospective cohort study. METHODS DNA samples from patients with normal-tension glaucoma and high-tension glaucoma and unaffected controls were screened for TBK1 copy number variations using real-time quantitative polymerase chain reaction. Samples with additional copies of the TBK1 gene were further tested using custom comparative genomic hybridization arrays. RESULTS Four out of 334 normal-tension glaucoma cases (1.2%) were found to carry TBK1 copy number variations using quantitative polymerase chain reaction. One extra dose of the TBK1 gene (duplication) was detected in 3 normal-tension glaucoma patients, while 2 extra doses of the gene (triplication) were detected in a fourth normal-tension glaucoma patient. The results were further confirmed by custom comparative genomic hybridization arrays. Further, the TBK1 copy number variation segregated with normal-tension glaucoma in the family members of the probands, showing an autosomal dominant pattern of inheritance. No TBK1 copy number variations were detected in 1045 Australian patients with high-tension glaucoma or in 254 unaffected controls. CONCLUSION We report the presence of TBK1 copy number variations in our Australian normal-tension glaucoma cohort, including the first example of more than 1 extra copy of this gene in glaucoma patients (gene triplication). These results confirm TBK1 to be an important cause of normal-tension glaucoma, but do not suggest common involvement in high-tension glaucoma. PMID:25284765

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1.

PubMed

Pan, Ling; Pasternak, David A; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W; Pan, Ying-Xian

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3' or 5' splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.

Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

PubMed

Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

2017-09-01

The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

Branched-chain amino acids in metabolic signalling and insulin resistance.

PubMed

Lynch, Christopher J; Adams, Sean H

2014-12-01

Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM.

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

PubMed

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Identification of a Key Gene Involved in Branched-Chain Short Fatty Acids Formation in Natto by Transcriptional Analysis and Enzymatic Characterization in Bacillus subtilis.

PubMed

Hong, Chenlu; Chen, Yangyang; Li, Lu; Chen, Shouwen; Wei, Xuetuan

2017-03-01

Natto as a fermented soybean product has many health benefits for human due to its rich nutritional and functional components. However, the unpleasant odor of natto, caused by the formation of branched-chain short fatty acids (BCFAs), prohibits the wide acceptance of natto products. This work is to identify the key gene of BCFAs formation and develop the guidance to reduce natto odor. Transcriptional analysis of BCFAs synthesis pathway genes was conducted in two Bacillus subtilis strains with obvious different BCFAs synthesis abilities. The transcriptional levels of bcd, bkdAA, and ptb in B. subtilis H-9 were 2.7-fold, 0.7-fold, and 8.9-fold higher than that of B. subtilis H-4, respectively. Therefore, the ptb gene with the highest transcriptional change was considered as the key gene in BCFAs synthesis. The ptb encoded enzyme Ptb was further characterized by inducible expression in Escherichia coli. The recombinant Ptb protein (about 32 kDa) was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The catalysis functions of Ptb were confirmed on substrates of isovaleryl-CoA and isobutyryl-CoA, and the higher catalysis efficiency of Ptb on isovaleryl-CoA explained the higher level of isovaleric acid in natto. The optimal activities of Ptb were observed at 50 °C and pH 8.0, and the enzymatic activity was inhibited by Ca 2+ , Zn 2+ , Ba 2+ , Mn 2+ , Cu 2+ , SDS, and EDTA. Collectively, this study reports a key gene responsible for BCFAs formation in natto fermentation and provides potential strategies to solve the odor problem.

A signature of six genes highlights defects on cell growth and specific metabolic pathways in murine and human hepatocellular carcinoma.

PubMed

Schröder, Paul C; Segura, Víctor; Riezu, José Ignacio; Sangro, Bruno; Mato, José M; Prieto, Jesús; Santamaría, Enrique; Corrales, Fernando J

2011-09-01

Hepatocellular carcinoma (HCC) represents a major health problem as it afflicts an increasing number of patients worldwide. Albeit most of the risk factors for HCC are known, this is a deadly syndrome with a life expectancy at the time of diagnosis of less than 1 year. Definition of the molecular principles governing the neoplastic transformation of the liver is an urgent need to facilitate the clinical management of patients, based on innovative methods to detect the disease in its early stages and on more efficient therapies. In the present study, we have combined the analysis of a murine model and human samples of HCC to identify genes differentially expressed early in the process of hepatocarcinogenesis, using a microarray-based approach. Expression of 190 genes was impaired in murine HCC from which 65 were further validated by low-density array real-time polymerase chain reaction (RT-PCR). The expression of the best 45 genes was then investigated in human samples resulting in 18 genes in which expression was significantly modified in HCC. Among them, JUN, methionine adenosyltransferase 1A and 2A, phosphoglucomutase 1, and acyl CoA dehydrogenase short/branched chain indicate defective cell proliferation as well as one carbon pathway, glucose and fatty acid metabolism, both in HCC and cirrhotic liver, a well-known preneoplastic condition. These alterations were further confirmed in public transcriptomic datasets from other authors. In addition, vasodilator-stimulated phosphoprotein, an actin-associated protein involved in cytoskeleton remodeling, was also found to be increased in the liver and serum of cirrhotic and HCC patients. In addition to revealing the impairment of central metabolic pathways for liver homeostasis, further studies may probe the potential value of the reported genes for the early detection of HCC.

Characterization of the 'Xiangshui' lemon transcriptome by de novo assembly to discover genes associated with self-incompatibility.

PubMed

Zhang, Shuwei; Ding, Feng; He, Xinhua; Luo, Cong; Huang, Guixiang; Hu, Ying

2015-02-01

Seedlessness is a desirable character in lemons and other citrus species. Seedless fruit can be induced in many ways, including through self-incompatibility (SI). SI is widely used as an intraspecific reproductive barrier that prevents self-fertilization in flowering plants. Although there have been many studies on SI, its mechanism remains unclear. The 'Xiangshui' lemon is an important seedless cultivar whose seedlessness has been caused by SI. It is essential to identify genes involved in SI in 'Xiangshui' lemon to clarify its molecular mechanism. In this study, candidate genes associated with SI were identified using high-throughput Illumina RNA sequencing (RNA-seq). A total of 61,224 unigenes were obtained (average, 948 bp; N50 of 1,457 bp), among which 47,260 unigenes were annotated by comparison to six public databases (Nr, Nt, Swiss-Prot, KEGG, COG, and GO). Differentially expressed genes were identified by comparing the transcriptomes of no-, self-, and cross-pollinated stigmas with styles of the 'Xiangshui' lemon. Several differentially expressed genes that might be associated with SI were identified, such as those involved in pollen tube growth, programmed cell death, signal transduction, and transcription. NADPH oxidase genes associated with apoptosis were highly upregulated in the self-pollinated transcriptome. The expression pattern of 12 genes was analyzed by quantitative real-time polymerase chain reaction. A putative S-RNase gene was identified that had not been previously associated with self-pollen rejection in lemon or citrus. This study provided a transcriptome dataset for further studies of SI and seedless lemon breeding.

Functional diversification of the dehydrin gene family in apple and its contribution to cold acclimation during dormancy.

PubMed

Falavigna, Vítor da Silveira; Miotto, Yohanna Evelyn; Porto, Diogo Denardi; Anzanello, Rafael; Santos, Henrique Pessoa dos; Fialho, Flávio Bello; Margis-Pinheiro, Márcia; Pasquali, Giancarlo; Revers, Luís Fernando

2015-11-01

Dehydrins (DHN) are proteins involved in plant adaptive responses to abiotic stresses, mainly dehydration. Several studies in perennial crops have linked bud dormancy progression, a process characterized by the inability to initiate growth from meristems under favorable conditions, with DHN gene expression. However, an in-depth characterization of DHNs during bud dormancy progression is still missing. An extensive in silico characterization of the apple DHN gene family was performed. Additionally, we used five different experiments that generated samples with different dormancy status, including genotypes with contrasting dormancy traits, to analyze how DHN genes are being regulated during bud dormancy progression in apple by real-time quantitative polymerase chain reaction (RT-qPCR). Duplication events took place in the diversification of apple DHN family. Additionally, MdDHN genes presented tissue- and bud dormant-specific expression patterns. Our results indicate that MdDHN genes are highly divergent in function, with overlapping levels, and that their expressions are fine-tuned by the environment during the dormancy process in apple. © 2015 Scandinavian Plant Physiology Society.

HOXB homeobox gene expression in cervical carcinoma.

PubMed

López, R; Garrido, E; Piña, P; Hidalgo, A; Lazos, M; Ochoa, R; Salcedo, M

2006-01-01

The homeobox (HOX) genes are a family of transcription factors that bind to specific DNA sequences in target genes regulating gene expression. Thirty-nine HOX genes have been mapped in four conserved clusters: A, B, C, and D; they act as master genes regulating the identity of body segments along the anteroposterior axis of the embryo. The role played by HOX genes in adult cell differentiation is unclear to date, but growing evidence suggests that they may play an important role in the development of cancer. To study the role played by HOX genes in cervical cancer, in the present work, we analyzed the expression of HOXB genes and the localization of their transcripts in human cervical tissues. Reverse transcription-polymerase chain reaction analysis and nonradioactive RNA in situ hybridization were used to detect HOXB expression in 11 normal cervical tissues and 17 cervical carcinomas. It was determined that HOXB1, B3, B5, B6, B7, B8, and B9 genes are expressed in normal adult cervical epithelium and squamous cervical carcinomas. Interestingly, HOXB2, HOXB4, and HOXB13 gene expression was found only in tumor tissues. Our findings suggest that the new expression of HOXB2, HOXB4, and B13 genes is involved in cervical cancer.

The altered liver microRNA profile in hepatotoxicity induced by rhizome Dioscorea bulbifera in mice.

PubMed

Yang, Rui; Bai, Qingyun; Zhang, Jiaqi; Sheng, Yuchen; Ji, Lili

2017-08-01

MicroRNA (miRNA) has been reported to play important roles in regulating drug-induced liver injury. Ethyl acetate extract isolated from rhizoma Dioscoreae bulbifera (EF) has been reported to induce hepatotoxicity in our previous studies. This study aims to observe the altered liver miRNA profile and its related signalling pathway involved in EF-induced hepatotoxicity. Serum alanine/aspartate aminotransferase assay showed that EF (450 mg/kg)-induced hepatotoxicity in mice. Results of miRNA chip analysis showed that the expression of eight miRNAs was up-regulated and of other nine miRNAs was down-regulated in livers from EF-treated mice. Further, the altered expression of miR-200a-3p, miR-5132-5p and miR-5130 was validated using real-time polymerase chain reaction (PCR) assay. There were total seven predicted target genes of miR-200a-3p, miR-5132-5p and miR-5130. Only one kyoto encyclopedia genes and genomes pathway was annotated using those target genes, which is protein processing in endoplasmic reticulum (ER). Furthermore, liver expression of DnaJ subfamily A member 1, a key gene involved in protein processing in ER based on the altered miRNAs, was increased in EF-treated mice. In conclusion, the results demonstrated that EF altered the expression of liver miRNA profile and its related signalling pathway, which may be involved in EF-induced hepatotoxicity.

A model for the evolution of the mammalian t-cell receptor α/δ and μ loci based on evidence from the duckbill Platypus.

PubMed

Parra, Zuly E; Lillie, Mette; Miller, Robert D

2012-10-01

The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRµ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRµ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRµ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRµ. The current structure of TCRδ and TCRµ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRµ locus.

A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus

PubMed Central

Parra, Zuly E.; Lillie, Mette; Miller, Robert D.

2012-01-01

The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRµ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRµ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRµ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRµ. The current structure of TCRδ and TCRµ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRµ locus. PMID:22593227

Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards.

PubMed

Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

2014-11-01

It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle.

Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards* #

PubMed Central

Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

2014-01-01

It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle. PMID:25367788

Identification and mapping of ts (tender spines), a gene involved in soft spine development in Cucumis sativus.

PubMed

Guo, Chunli; Yang, Xuqin; Wang, Yunli; Nie, Jingtao; Yang, Yi; Sun, Jingxian; Du, Hui; Zhu, Wenying; Pan, Jian; Chen, Yue; Lv, Duo; He, Huanle; Lian, Hongli; Pan, Junsong; Cai, Run

2018-01-01

Using map-based cloning of ts gene, we identified a new sort of gene involved in the initiation of multicellular tender spine in cucumber. The cucumber (Cucumis sativus L.) fruit contains spines on the surface, which is an extremely valuable quality trait affecting the selection of customers. In this study, we elaborated cucumber line NC072 with wild type (WT) hard fruit spines and its spontaneous mutant NC073, possessing tender and soft spines on fruits. The mutant trait was named as tender spines (ts), which is controlled by a single recessive nuclear gene. We identified the gene ts by map-based cloning with an F 2 segregating population of 721 individuals generated from NC073 and WT line SA419-2. It was located between two markers Indel6239679 and Indel6349344, 109.7 kb physical distance on chromosome 1 containing fifteen putative genes. With sequencing and quantitative reverse transcription-polymerase chain reaction analysis, the Csa1G056960 gene was considered as the most possible candidate gene of ts. In the mutant, Csa1G056960 has a nucleotide change in the 5' splicing site of the second intron, which causes different splicing to delete the second exon, resulting in a N-terminal deletion in the predicted amino acid sequence. The gene encodes a C-type lectin receptor-like tyrosine-protein kinase which would play an important role in the formation of cucumber fruit. This is firstly reported of a receptor kinase gene regulating the development of multicellular spines/trichomes in plants. The ts allele could accelerate the molecular breeding of cucumber soft spines.

Interleukin-5 regulates genes involved in B-cell terminal maturation.

PubMed

Horikawa, Keisuke; Takatsu, Kiyoshi

2006-08-01

Interleukin (IL)-5 induces CD38-activated splenic B cells to differentiate into immunoglobulin M-secreting cells and undergo micro to gamma 1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL-4, a well-known IgG1-inducing factor does not induce immunoglobulin production or micro to gamma 1 CSR in CD38-activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL-5-induced gene expression in CD38-stimulated B cells to immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR. IL-5 and IL-4 stimulation of CD38-activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL-5-inducible 78 genes were redundantly regulated by IL-4. IL-5 and IL-4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL-5 include immunoglobulin-related genes such as J chain and immunoglobulinkappa, and genes involved in B-cell maturation such as BCL6, activation-induced cytidine deaminase (Aid) and B lymphocyte-induced maturation protein-1 (Blimp-1) and tend to be induced slowly after IL-5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp-1 and Aid in CD38-activated B cells could induce IL-4-dependent maturation to Syndecan-1+ antibody-secreting cells and micro to gamma 1 CSR, respectively, in CD38-activated B cells. Taken together, preferential Aid and Blimp-1 expression plays a critical role in IL-5-induced immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR in CD38-activated B cells.

Comprehensive Gene Expression Analysis of Rice Aleurone Cells: Probing the Existence of an Alternative Gibberellin Receptor1

PubMed Central

Yano, Kenji; Aya, Koichiro; Hirano, Ko; Ordonio, Reynante Lacsamana; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2015-01-01

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells. PMID:25511432

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Characterization of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in Shanghai, China: molecular and cytogenetic characteristics, IgV gene restriction and hypermutation patterns.

PubMed

Irons, Richard D; Le, Anh; Bao, Liming; Zhu, Xiongzeng; Ryder, John; Wang, Xiao Qin; Ji, Meirong; Chen, Yan; Wu, Xichun; Lin, Guowei

2009-12-01

The clinical, cytogenetic and molecular features of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a disease previously considered to be rare in Asia, were examined in consecutive series of 70 cases diagnosed by our laboratory over a 30-month period. Clonal abnormalities were observed in 80% of CLL/SLL cases using a combination of conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis. Those involving 14q32/IGH were the most frequent (24 cases), followed by trisomy 12 and 11q abnormalities. IgV(H) gene usage was non-random with over-representation of V(H)4-34, V(H)3-23 and a previously unreported increase in V(H)3-48 gene use. Somatic hypermutation (SHM) of IgV(H) germline sequences was observed in 56.5% of cases with stereotyped patterns of SHM observed in V(H)4-34 heavy chain complimentary-determining (HCDR1) and framework region CFR2 sequences. These findings in a Chinese population suggest subtle geographical differences in IgV(H) gene usage while the remarkably specific pattern of SHM suggest that a relatively limited set of antigens may be involved in the development of this disease worldwide. IgV(H) gene mutation status was a significant predictor of initial survival in CLL/SLL. However, an influence of karyotype on prognosis was not observed.

High Variation in Pathogenicity of Genetically Closely Related Strains of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen, in Guadeloupe.

PubMed

Champoiseau, P; Daugrois, J-H; Pieretti, I; Cociancich, S; Royer, M; Rott, P

2006-10-01

ABSTRACT Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.

Association of interleukin-10 gene polymorphisms with breast cancer in a Chinese population.

PubMed

Kong, Fanjun; Liu, Jie; Liu, Yongheng; Song, Bao; Wang, Hualing; Liu, Wenchao

2010-06-17

Interleukin-10(IL-10) is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions. Polymorphisms in the IL-10 gene promoter genetically determine interindividual differences in IL-10 production. This study was performed to determined whether polymorphisms in the IL-10 gene promoter were associated with breast cancer in a Chinese Han population. We genotyped 315 patients with breast cancer and 322 healthy control subjects for -1082A/G, -819T/C and -592A/C single nucleotide polymorphisms in the promoter region of the IL-10 gene by polymerase chain reactionerestriction fragment length polymorphism (PCR-RFLP). There were no significant differences in genotype, allele, or haplotype frequencies in all three loci between patients and healthy controls. Analysis of breast cancer prognostic and predictive factors revealed that the -1082AA genotype was associated with a significantly increased risk of lymph node (LN) involvement (P = 0.041) and larger tumor size (P = 0.039) at the time of diagnosis. Furthermore, in the haplotype analysis of IL-10 gene, we found that patients carrying ATA haplotype were in higher LN involvement (p = 0.022) and higher tumor stage(p = 0.028) of breast cancer at the time of diagnosis compared with others. Our findings suggest that IL-10 promoter polymorphisms participate in the progression of breast cancer rather than in its initial development in Chinese Han women.

Influence of rice whole-crop silage diet on growth performance, carcass and meat characteristics and muscle-related gene expression in Japanese Black steers.

PubMed

Shibata, Masahiro; Hikino, Yasuko; Imanari, Mai; Matsumoto, Kazunori; Yamamoto, Naoyuki

2016-07-01

The present study investigated the influence of a diet largely comprising rice whole-crop silage (rWCS) on growth performance, carcass and meat characteristics, and expression of genes involved in muscle growth of Japanese Black steers. Steers were randomly separated into rWCS-fed (rWCS ad libitum and restricted feeding of concentrate) and concentrate-fed groups. Total digestible nutrient intake and daily gain (DG) decreased in rWCS-fed steers in comparison with concentrate-fed steers, whereas dressed carcass weight and final body weight did not significantly differ between the groups. Decreases in drip loss in the muscle of rWCS-fed steers may be caused by α-tocopherol and β-carotene in muscle. Feeding large amounts of rWCS to steers may maintain quantitative productivity of beef steers equally to a concentrate-based diet, and improve the qualitative productivity. Results of gene expression suggest that activation of skeletal muscle growth in rWCS-fed steers may occur at the late fattening period owing to a decrease in myostatin and increase in myosin heavy chain gene expression. Preadipocyte factor-1 and myostatin genes may be strongly involved in the control of lipid accumulation. This rearing system would allow beef production to switch to rWCS-based diets from concentrate-based diets. © 2015 Japanese Society of Animal Science.

Mapping of a Cellulose-Deficient Mutant Named dwarf1-1 in Sorghum bicolor to the Green Revolution Gene gibberellin20-oxidase Reveals a Positive Regulatory Association between Gibberellin and Cellulose Biosynthesis.

PubMed

Petti, Carloalberto; Hirano, Ko; Stork, Jozsef; DeBolt, Seth

2015-09-01

Here, we show a mechanism for expansion regulation through mutations in the green revolution gene gibberellin20 (GA20)-oxidase and show that GAs control biosynthesis of the plants main structural polymer cellulose. Within a 12,000 mutagenized Sorghum bicolor plant population, we identified a single cellulose-deficient and male gametophyte-dysfunctional mutant named dwarf1-1 (dwf1-1). Through the Sorghum propinquum male/dwf1-1 female F2 population, we mapped dwf1-1 to a frameshift in GA20-oxidase. Assessment of GAs in dwf1-1 revealed ablation of GA. GA ablation was antagonistic to the expression of three specific cellulose synthase genes resulting in cellulose deficiency and growth dwarfism, which were complemented by exogenous bioactive gibberellic acid application. Using quantitative polymerase chain reaction, we found that GA was positively regulating the expression of a subset of specific cellulose synthase genes. To cross reference data from our mapped Sorghum sp. allele with another monocotyledonous plant, a series of rice (Oryza sativa) mutants involved in GA biosynthesis and signaling were isolated, and these too displayed cellulose deficit. Taken together, data support a model whereby suppressed expansion in green revolution GA genes involves regulation of cellulose biosynthesis. © 2015 American Society of Plant Biologists. All Rights Reserved.

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

PubMed

Parvari, R; Avivi, A; Lentner, F; Ziv, E; Tel-Or, S; Burstein, Y; Schechter, I

1988-03-01

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.

When Genome-Based Approach Meets the “Old but Good”: Revealing Genes Involved in the Antibacterial Activity of Pseudomonas sp. P482 against Soft Rot Pathogens

PubMed Central

Krzyżanowska, Dorota M.; Ossowicki, Adam; Rajewska, Magdalena; Maciąg, Tomasz; Jabłońska, Magdalena; Obuchowski, Michał; Heeb, Stephan; Jafra, Sylwia

2016-01-01

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYST. Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis. PMID:27303376

Chromosomal rearrangement of the PAX-5 locus in lymphoplasmacytic lymphoma with t(9;14)(p13;q32).

PubMed

Iida, S; Rao, P H; Ueda, R; Chaganti, R S; Dalla-Favera, R

1999-06-01

B-cell non-Hodgkin's lymphoma (NHL) consists of heterogeneous subtypes based on histologic, immunophenotypic, and clinical findings. Recent advances in molecular biology have provided us new insights into the pathogenesis of this neoplasm at the genetic level, such as the deregulation of the protooncogenes adjoining the immunoglobulin gene (Ig) loci, which is a specific event in mature B-cell tumors. Moreover, involvement of certain protooncogenes corresponds to certain subtypes of NHL. Recently, we found that t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytic lymphoma (LPL) juxtaposes PAX-5 gene encoding for an essential transcription factor (BSAP: B-cell specific activator protein) for B-cell proliferation and differentiation to the Ig heavy chain gene (IgH) locus. This results in deregulated expression of the PAX-5 mRNA. We also developed a diagnostic FISH (fluorescence in situ hybridization) procedure which is able to detect 80% of the widely scattering 9p13 breakpoints involved in this translocation. Thus, an understanding of the PAX-5 gene's physiological role in B-cell development and the pathological role in tumorigenesis may lead to the optimal clinical treatment strategy for LPL and LPL-derived diffuse large cell lymphoma (DLCL).

Identification and expression analysis of genes involved in early ovary development in diploid gynogenetic hybrids of red crucian carp x common carp.

PubMed

Liu, Dong; Liu, Shaojun; You, Cuiping; Chen, Lin; Liu, Zhen; Liu, Liangguo; Wang, Jing; Liu, Yun

2010-04-01

Diploid eggs of allotetraploid hybrids (red crucian carp female symbol x common carp male symbol), when activated by UV-irradiated sperm of scatter scale carp, can develop into diploid progenies without chromosome duplication treatment. Diploid progenies produce diploid eggs, which develop into diploid population by the same way. To understand the molecular mechanism underlying the production of diploid eggs by the diploid fish, we constructed a forward suppression subtractive hybridization complementary DNA (cDNA) library. The cDNAs from the ovary in proliferation phase were employed as the "tester," and those in growth phase were used as the "driver." Seventy-three cDNA clones that are specifically expressed in proliferation phase were detected by dot-blot hybridization. Sequencing analyses revealed that several of these cDNAs have high homologies to the known sequences in the NCBI database. Their encoded proteins include the protein preventing mitosis catastrophe (PMC), the signal recognition particle 9, the ATP-binding cassette transporter, the glucanase-xylanase fusion protein, and others. These genes were confirmed by reverse transcriptase-polymerase chain reaction. The expression profile of the PMC gene at different time points was analyzed by quantitative real-time polymerase chain reaction. The results indicated that the expression of this suppression subtractive hybridization-identified gene changed during the time course, corresponding with the cellular phenomenon in the ovary development. Our studies provide insights into the molecular mechanism underlying the ovary development of diploid gynogenetic fish.

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

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

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

The unique sex chromosome system in platypus and echidna.

PubMed

Ferguson-Smith, M A; Rens, W

2010-10-01

A striking example of the power of chromosome painting has been the resolution of the male platypus karyotype and the pairing relationships of the chain often sex chromosomes. We have extended our analysis to the nine sex chromosomes of the male echidna. Cross-species painting with platypus shows that the first five chromosomes in the chain are identical in both, but the order of the remainder are different and, in each species, a different autosome replaces one of the five X chromosomes. As the therian X is homologous mainly to platypus autosome 6 and echidna 16, and as SRY is absent in both, the sex determination mechanism in monotremes is currently unknown. Several of the X and Y chromosomes contain genes orthologous to those in the avian Z but the significance of this is also unknown. It seems likely that a novel testis determinant is carried by a Y chromosome common to platypus and echidna. We have searched for candidates for this determinant among the many genes known to be involved in vertebrate sex differentiation. So far fourteen such genes have been mapped, eleven are autosomal in platypus, two map to the differential regions of X chromosomes, and one maps to a pairing segment and is likewise excluded. Search for the platypus testis-determining gene continues, and the extension of comparative mapping between platypus and birds and reptiles may shed light on the ancestral origin of monotreme sex chromosomes.

The extent to which immunity, apoptosis and detoxification gene expression interact with 17 alpha-methyltestosterone.

PubMed

Abo-Al-Ela, Haitham G; El-Nahas, Abeer F; Mahmoud, Shawky; Ibrahim, Essam M

2017-01-01

Innate immunity is the first line of defence against invasion by foreign pathogens. One widely used synthetic androgen for the production of all-male fish, particularly commercially valuable Nile tilapia, Oreochromis niloticus, is 17 alpha-methyltestosterone (MT). The present study investigates the effect of MT on innate immunity, cellular apoptosis and detoxification and the mortality rate, during and after the feeding of fry with 0-, 40-and 60-mg MT/kg. Expression analysis was completed on interleukin 1 beta (il1β), interleukin 8 (il8), tumour necrosis factor alpha (tnfα), CXC2- and CC-chemokines, interferon (ifn), myxovirus resistance (mx), toll-like receptor 7 (tlr7), immunoglobulin M heavy chain (IgM heavy chain), vitellogenin (vtg), cellular apoptosis susceptibility (cas) and glutathione S-transferase α1 (gstα1). Expression analysis revealed that MT had a significant impact on these genes, and this impact varied from induction to repression during and after the treatment. Linear regression analysis showed a significant association between the majority of the tested gene transcript levels and mortality rates on the 7 th and 21 st days of hormonal treatment and 2 weeks following hormonal cessation. The results are thoroughly discussed in this article. This is the first report concerning the hazardous effect of MT on a series of genes involved in immunity, apoptosis and detoxification in the Nile tilapia fry. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel large deletion mutation of FERMT1 gene in a Chinese patient with Kindler syndrome.

PubMed

Gao, Ying; Bai, Jin-li; Liu, Xiao-yan; Qu, Yu-jin; Cao, Yan-yan; Wang, Jian-cai; Jin, Yu-wei; Wang, Hong; Song, Fang

2015-11-01

Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive skin disorder, which results in symptoms including blistering, epidermal atrophy, increased risk of cancer, and poor wound healing. The majority of mutations of the disease-determining gene (FERMT1 gene) are single nucleotide substitutions, including missense mutations, nonsense mutations, etc. Large deletion mutations are seldom reported. To determine the mutation in the FERMT1 gene associated with a 7-year-old Chinese patient who presented clinical manifestation of KS, we performed direct sequencing of all the exons of FERMT1 gene. For the exons 2-6 without amplicons, we analyzed the copy numbers using quantitative real-time polymerase chain reaction (qRT-PCR) with specific primers. The deletion breakpoints were sublocalized and the range of deletion was confirmed by PCR and direct sequencing. In this study, we identified a new 17-kb deletion mutation spanning the introns 1-6 of FERMT1 gene in a Chinese patient with severe KS phenotypes. Her parents were carriers of the same mutation. Our study reported a newly identified large deletion mutation of FERMT1 gene involved in KS, which further enriched the mutation spectrum of the FERMT1 gene.

Homologues of CsLOB1 in citrus function as disease susceptibility genes in citrus canker.

PubMed

Zhang, Junli; Huguet-Tapia, Jose Carlos; Hu, Yang; Jones, Jeffrey; Wang, Nian; Liu, Sanzhen; White, Frank F

2017-08-01

The lateral organ boundary domain (LBD) genes encode a group of plant-specific proteins that function as transcription factors in the regulation of plant growth and development. Citrus sinensis lateral organ boundary 1 (CsLOB1) is a member of the LBD family and functions as a disease susceptibility gene in citrus bacterial canker (CBC). Thirty-four LBD members have been identified from the Citrus sinensis genome. We assessed the potential for additional members of LBD genes in citrus to function as surrogates for CsLOB1 in CBC, and compared host gene expression on induction of different LBD genes. Using custom-designed transcription activator-like (TAL) effectors, two members of the same clade as CsLOB1, named CsLOB2 and CsLOB3, were found to be capable of functioning similarly to CsLOB1 in CBC. RNA sequencing and quantitative reverse transcription-polymerase chain reaction analyses revealed a set of cell wall metabolic genes that are associated with CsLOB1, CsLOB2 and CsLOB3 expression and may represent downstream genes involved in CBC. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Paradoxical effect of mitochondrial respiratory chain impairment on insulin signaling and glucose transport in adipose cells.

PubMed

Shi, Xiarong; Burkart, Alison; Nicoloro, Sarah M; Czech, Michael P; Straubhaar, Juerg; Corvera, Silvia

2008-11-07

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family.

PubMed

Tan, J; Tay, J S; Wong, Y C; Kham, S K; Bte Abd Aziz, N; Teo, S H; Wong, H B

1995-01-01

Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/-alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q-chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E.

Genes encoding Xenopus laevis Ig L chains: Implications for the evolution of [kappa] and [lambda] chains

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

Zezza, D.J.; Stewart, S.E.; Steiner, L.A.

1992-12-15

Xenopus laevis Ig contain two distinct types of L chains, designated [rho] or L1 and [sigma] or L2. The authors have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and Cmore » gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian [kappa] chains than to those of mammalian [lambda] chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J[kappa]. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V[kappa]. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5[prime]-flanking region, but diverges in sequence 5[prime] to position [minus]95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3[prime]-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3[prime]-ends, as in V[kappa]. Taken together, the data suggest that Xenopus L1 L chain genes are members of the [kappa] gene family. 80 refs., 9 figs.« less

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

PubMed

Kihara, Takahiro; Hiroe, Ayaka; Ishii-Hyakutake, Manami; Mizuno, Kouhei; Tsuge, Takeharu

2017-08-01

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJ YB4 ) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJ YB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJ YB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJ YB4 , which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJ YB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants.

PubMed

Yang, Chunxiao; Preisser, Evan L; Zhang, Hongjun; Liu, Yong; Dai, Liangying; Pan, Huipeng; Zhou, Xuguo

2016-01-01

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata , a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper , the Δ C t method, and RefFinder , were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata , our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

PubMed

Dunnick, Wesley A; Shi, Jian; Holden, Victoria; Fontaine, Clinton; Collins, John T

2011-01-01

Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is <1% the wild-type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

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

Vandenberg, P.; Khillan, J.S.; Prockop, D.J.

A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing themore » minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.« less

Hereditary sensory and autonomic neuropathy type I in a Chinese family: British C133W mutation exists in the Chinese.

PubMed

Bi, Hongyan; Gao, Yunying; Yao, Sheng; Dong, Mingrui; Headley, Alexander Peter; Yuan, Yun

2007-10-01

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disorder of the peripheral nervous system characterized by marked progressive sensory loss, with variable autonomic and motor involvement. The HSAN I locus maps to chromosome 9q22.1-22.3 and is caused by mutations in the gene coding for serine palmitoyltransferase long chain base subunit 1 (SPTLC1). Sequencing in HSAN I families have previously identified mutations in exons 5, 6 and 13 of this gene. Here we report the clinical, electrophysiological and pathological findings of a proband in a Chinese family with HSAN I. The affected members showed almost typical clinical features. Electrophysiological findings showed an axonal, predominantly sensory, neuropathy with motor and autonomic involvement. Sural nerve biopsy showed loss of myelinated and unmyelinated fibers. SPTLC1 mutational analysis revealed the C133W mutation, a mutation common in British HSAN I families.

Hearing Loss in Osteogenesis Imperfecta: Characteristics and Treatment Considerations

PubMed Central

Pillion, Joseph P.; Vernick, David; Shapiro, Jay

2011-01-01

Osteogenesis imperfecta (OI) is the most common heritable disorder of connective tissue. It is associated with fractures following relatively minor injury, blue sclerae, dentinogenesis imperfecta, increased joint mobility, short stature, and hearing loss. Structures in the otic capsule and inner ear share in the histologic features common to other skeletal tissues. OI is due to mutations involving several genes, the most commonly involved are the COL1A1 or COL1A2 genes which are responsible for the synthesis of the proalpha-1 and proalpha-2 polypeptide chains that form the type I collagen triple helix. A genotype/phenotype relationship to hearing loss has not been established in OI. Hearing loss is commonly found in OI with prevalence rates ranging from 50 to 92% in some studies. Hearing loss in OI may be conductive, mixed, or sensorineural and is more common by the second or third decade. Treatment options such as hearing aids, stapes surgery, and cochlear implants are discussed. PMID:22567374

Microbial communities involved in methane production from hydrocarbons in oil sands tailings.

PubMed

Siddique, Tariq; Penner, Tara; Klassen, Jonathan; Nesbø, Camilla; Foght, Julia M

2012-09-04

Microbial metabolism of residual hydrocarbons, primarily short-chain n-alkanes and certain monoaromatic hydrocarbons, in oil sands tailings ponds produces large volumes of CH(4) in situ. We characterized the microbial communities involved in methanogenic biodegradation of whole naphtha (a bitumen extraction solvent) and its short-chain n-alkane (C(6)-C(10)) and BTEX (benzene, toluene, ethylbenzene, and xylenes) components using primary enrichment cultures derived from oil sands tailings. Clone libraries of bacterial 16S rRNA genes amplified from these enrichments showed increased proportions of two orders of Bacteria: Clostridiales and Syntrophobacterales, with Desulfotomaculum and Syntrophus/Smithella as the closest named relatives, respectively. In parallel archaeal clone libraries, sequences affiliated with cultivated acetoclastic methanogens (Methanosaetaceae) were enriched in cultures amended with n-alkanes, whereas hydrogenotrophic methanogens (Methanomicrobiales) were enriched with BTEX. Naphtha-amended cultures harbored a blend of these two archaeal communities. The results imply syntrophic oxidation of hydrocarbons in oil sands tailings, with the activities of different carbon flow pathways to CH(4) being influenced by the primary hydrocarbon substrate. These results have implications for predicting greenhouse gas emissions from oil sands tailings repositories.

Molecular characterization and immunological response analysis of a novel transferrin-like, pacifastin heavy chain protein in giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879).

PubMed

Toe, Aung; Areechon, Nontawith; Srisapoome, Prapansak

2012-10-01

The full-length cDNA of the pacifastin heavy chain gene from giant freshwater prawn (Macrobrachium rosenbergii, Mr-PHC) was cloned and characterized. The full sequence of the Mr-PHC cDNA was 4331 bp and contained a 119-bp 5'-untranslated region (UTR), a 3990-bp open reading frame (ORF) encoding 1329 amino acid residues and a 222-bp 3' UTR. The Mr-PHC protein predicted by its full ORF, exhibited a unique transferrin-like protein structure containing 4 different lobes that have not been previously identified. Three of the four lobes contained highly conserved of iron/anion binding residues. Expression analyses by conventional RT-PCR demonstrated that Mr-PHC was expressed predominantly during postlarval stage 45 and also in the foregut and gills of the adult prawn. Interestingly, dose response analyses that were quantified using quantitative real-time PCR indicated a significant upregulation of Mr-PHC during postlarval stage 45 in prawn grown at hour 24 after challenging with 10(9) cfu/ml of Aeromonas hydrophila, which is a pathogenic bacterium. Mr-HPC in the adult prawn was significantly upregulated at both hour 12 and day 7 after stimulation with A. hydrophila (P < 0.05 and P < 0.01, respectively). Additionally, a delayed induction response of the Mr-PHC gene was observed at 14 days when the experimental adult prawns were fed with β-glucan-supplemented feed. Based on results of this study, the transferrin-like protein encoded by the pacifastin heavy chain gene may exist in all decapod crustaceans. Even though the function as an iron transporter is not proven, immune response studies are clearly indicated that PHC is critically involved in the immune system in these animals.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

PubMed

Kruse, Thomas; van de Pas, Bram A; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M; van der Oost, John; Smidt, Hauke; Stams, Alfons J M

2015-03-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

PubMed Central

van de Pas, Bram A.; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R.; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M.; van der Oost, John; Smidt, Hauke

2014-01-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. PMID:25512312

Relationships between the transcriptome and physiological indicators of reproduction in female rainbow trout over an annual cycle.

PubMed

Hook, Sharon E; Nagler, James J; Cavileer, Tim; Verducci, Joseph; Liu, Yushi; Hayton, William; Schultz, Irvin R

2011-02-01

Normal transcriptomic patterns along the brain-pituitary-gonad-liver (BPGL) axis should be better characterized if endocrine-disrupting compound-induced changes in gene expression are to be understood. Female rainbow trout were studied over a complete year-long reproductive cycle. Tissue samples from pituitary, ovary, and liver were collected for microarray analysis using the 16K Genomic Research on Atlantic Salmon Project (GRASP) microarray and for quantitative polymerase chain reaction measures of estrogen receptor (ER) isoform messenger RNA (mRNA) levels. Plasma was collected to determine levels of circulating estradiol-17β (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). As an a priori hypothesis, changes in gene expression were correlated to either circulating levels of E2, FSH, and LH, or ER mRNAs quantified by quantitative polymerase chain reaction. In the liver, most transcriptomic patterns correlated to levels of either E2, LH, or ERs. Fewer ovarian transcripts could be correlated to levels of E2, ERα, or FSH. No significant associations were obvious in the pituitary. As a post hoc hypothesis, changes in transcript abundance were compared with microarray features with known roles in gonadal maturation. Many altered transcripts in the ovary correlated to transcript levels of estradiol 17-beta-dehydrogenase 8 or 17 B HSD12, or to glycoprotein alpha chain 1 or 2. In the pituitary, genes involved with the growth axis (e.g., growth hormone, insulin-related growth factor binding protein) correlated with the most transcripts. These results suggest that transcriptional networks along the BPGL axis may be regulated by factors other than circulating steroid hormones. © 2010 SETAC.

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

PubMed

Sirey, Tamara M; Ponting, Chris P

2016-10-15

The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

Transfer of the α5(IV) Collagen Chain Gene to Smooth Muscle Restores in Vivo Expression of the α6(IV) Collagen Chain in a Canine Model of Alport Syndrome

PubMed Central

Harvey, Scott J.; Zheng, Keqin; Jefferson, Barbara; Moak, Peter; Sado, Yoshikazu; Naito, Ichiro; Ninomiya, Yoshifumi; Jacobs, Robert; Thorner, Paul S.

2003-01-01

X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome. PMID:12598321

Posttransplant lymphoproliferative disorder in a kidney-pancreas transplanted recipient: simultaneous development of clonal lymphoid B-cell proliferation of host and donor origin.

PubMed

Heyny-von Haussen, Roland; Klingel, Karin; Riegel, Werner; Kandolf, Reinhard; Mall, Gerhard

2006-07-01

Posttransplant lymphoproliferative disorders (PTLDs) are lymphoid proliferations or lymphomas that develop as a consequence of immunosuppression after solid organ or bone marrow transplantation and are mostly associated with an Epstein-Barr virus infection. The morphologic categories include different types of benign and malignant lymphoid proliferations. The majority of PTLDs is of B-cell origin with clonal rearrangements of the immunoglobulin genes. The PTLDs in solid organ transplants are reported to be either of host or of donor origin. Donor-related PTLDs frequently involve the allograft. We report a case of a 52-year-old woman recipient who developed simultaneously PTLDs in several organs 5 month after receiving a sex-mismatched renal and pancreas allograft. Immunosuppression regimen comprised antithymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids. Pathologic features appeared as polymorphic PTLDs in the renal allograft, liver, and central nervous system (CNS). Molecular genetic studies revealed different clonal immunoglobulin heavy chain gene rearrangements in all 3 organs as determined by polymerase chain reaction (PCR). Epstein-Barr virus were detected by nested PCR and in situ hybridization in all 3 tumors. The PTLDs in liver and CNS were of host origin whereas the allograft kidney PTLD was found to originate from the male donor as shown by the simultaneous detection of female and male sex chromosomes by PCR and fluorescence in situ hybridization. The recipient died in consequence of the CNS involvement, after intracerebral hemorrhage with uncal and tonsillar herniation.

Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

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

Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu

Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed tomore » differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.« less

Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification.

PubMed

Ohtake, A; Murayama, K; Mori, M; Harashima, H; Yamazaki, T; Tamaru, S; Yamashita, Y; Kishita, Y; Nakachi, Y; Kohda, M; Tokuzawa, Y; Mizuno, Y; Moriyama, Y; Kato, H; Okazaki, Y

2014-04-01

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

PubMed

Lundqvist, Mats L; Kohlberg, Kathleen E; Gefroh, Holly A; Arnaud, Philippe; Middleton, Darlene L; Romano, Tracy A; Warr, Gregory W

2002-07-01

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.

De Novo Characterization of the Spleen Transcriptome of the Large Yellow Croaker (Pseudosciaena crocea) and Analysis of the Immune Relevant Genes and Pathways Involved in the Antiviral Response

PubMed Central

Ding, Yang; Ao, Jingqun; Hu, Songnian; Chen, Xinhua

2014-01-01

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. To understand the molecular basis for antiviral defense in this species, we used Illumia paired-end sequencing to characterize the spleen transcriptome of polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced large yellow croakers. The library produced 56,355,728 reads and assembled into 108,237 contigs. As a result, 15,192 unigenes were found from this transcriptome. Gene ontology analysis showed that 4,759 genes were involved in three major functional categories: biological process, cellular component, and molecular function. We further ascertained that numerous consensus sequences were homologous to known immune-relevant genes. Kyoto Encyclopedia of Genes and Genomes orthology mapping annotated 5,389 unigenes and identified numerous immune-relevant pathways. These immune-relevant genes and pathways revealed major antiviral immunity effectors, including but not limited to: pattern recognition receptors, adaptors and signal transducers, the interferons and interferon-stimulated genes, inflammatory cytokines and receptors, complement components, and B-cell and T-cell antigen activation molecules. Moreover, the partial genes of Toll-like receptor signaling pathway, RIG-I-like receptors signaling pathway, Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, and T-cell receptor (TCR) signaling pathway were found to be changed after poly(I:C) induction by real-time polymerase chain reaction (PCR) analysis, suggesting that these signaling pathways may be regulated by poly(I:C), a viral mimic. Overall, the antivirus-related genes and signaling pathways that were identified in response to poly(I:C) challenge provide valuable leads for further investigation of the antiviral defense mechanism in the large yellow croaker. PMID:24820969

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L.

PubMed

Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; van Dijk, Jeroen P; Kok, Esther J; Frazzon, Jeverson

2016-02-03

Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

Characterisation of the Transcriptomes of Genetically Diverse Listeria monocytogenes Exposed to Hyperosmotic and Low Temperature Conditions Reveal Global Stress-Adaptation Mechanisms

PubMed Central

Durack, Juliana; Ross, Tom; Bowman, John P.

2013-01-01

The ability of Listeria monocytogenes to adapt to various food and food- processing environments has been attributed to its robustness, persistence and prevalence in the food supply chain. To improve the present understanding of molecular mechanisms involved in hyperosmotic and low-temperature stress adaptation of L. monocytogenes, we undertook transcriptomics analysis on three strains adapted to sub-lethal levels of these stress stimuli and assessed functional gene response. Adaptation to hyperosmotic and cold-temperature stress has revealed many parallels in terms of gene expression profiles in strains possessing different levels of stress tolerance. Gene sets associated with ribosomes and translation, transcription, cell division as well as fatty acid biosynthesis and peptide transport showed activation in cells adapted to either cold or hyperosmotic stress. Repression of genes associated with carbohydrate metabolism and transport as well as flagella was evident in stressed cells, likely linked to activation of CodY regulon and consequential cellular energy conservation. PMID:24023890

Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.

PubMed

Suárez-Castillo, Edna C; Medina-Ortíz, Wanda E; Roig-López, José L; García-Arrarás, José E

2004-06-09

We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules.

Characterization of an Arxula adeninivorans alcohol dehydrogenase involved in the metabolism of ethanol and 1-butanol.

PubMed

Kasprzak, Jakub; Rauter, Marion; Riechen, Jan; Worch, Sebastian; Baronian, Kim; Bode, Rüdiger; Schauer, Frieder; Kunze, Gotthard

2016-05-01

In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Differential gene expression in migratory streams of cortical interneurons

PubMed Central

Antypa, Mary; Faux, Clare; Eichele, Gregor; Parnavelas, John G; Andrews, William D

2011-01-01

Cortical interneurons originate in the ganglionic eminences of the subpallium and migrate into the cortex in well-defined tangential streams. At the start of corticogenesis, two streams of migrating neurons are evident: a superficial one at the level of the preplate (PPL), and a deeper one at the level of the intermediate zone (IZ). Currently, little is known about the signalling mechanisms that regulate interneuron migration, and almost nothing is known about the molecules that may be involved in their choice of migratory stream. Here, we performed a microarray analysis, comparing the changes in gene expression between cells migrating in the PPL and those migrating in the IZ at embryonic day 13.5. This analysis identified genes, many of them novel, that were upregulated in one of the two streams. Moreover, polymerase chain reaction, in situ hybridization experiments and immunohistochemistry showed the expression of these genes in interneurons migrating within the PPL or IZ, suggesting that they play a role in their migration and choice of stream. PMID:22103416

Polymorphisms in NAT2 (N-acetyltransferase 2) gene in patients with systemic lupus erythematosus.

PubMed

Santos, Elaine Cristina Lima Dos; Pinto, Amanda Chaves; Klumb, Evandro Mendes; Macedo, Jacyara Maria Brito

To investigate potential associations of four substitutions in NAT2 gene and of acetylator phenotype of NAT2 with systemic lupus erythematosus (SLE) and clinical phenotypes. Molecular analysis of 481C>T, 590G>A, 857G>A, and 191G>A substitutions in the NAT2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, from DNA extracted from peripheral blood samples obtained from patients with SLE (n=91) and controls (n=97). The 857GA genotype was more prevalent among nonwhite SLE patients (OR=4.01, 95% CI=1.18-13.59). The 481T allele showed a positive association with hematological disorders that involve autoimmune mechanisms, specifically autoimmune hemolytic anemia or autoimmune thrombocytopenia (OR=1.97; 95% CI=1.01-3.81). Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

A more accurate detection of codon 72 polymorphism and LOH of the TP53 gene.

PubMed

Baccouche, Sami; Mabrouk, Imed; Said, Salem; Mosbah, Ali; Jlidi, Rachid; Gargouri, Ali

2003-01-10

The polymorphism at codon 72 of the TP53 gene has been extensively studied for its involvement in cancerogenesis and loss of heterozygosity (LOH) detection. Usually, the exon 4 of the TP53 gene is amplified by polymerase chain reaction (PCR) on DNA extracted from blood and tumor tissues, then digested by AccII. In the case of heterozygosity, the comparison of AccII profile from blood and tumor DNA PCR products allowed the identification of a potential LOH in the TP53 locus. This method can be hindered by a partial AccII digestion and/or DNA contamination of non-tumor cells. To circumvent these problems, we have developed a new approach by using the AccII restriction site between exon 4 and exon 6. The PCR amplification of exon 4-6, followed by AccII digestion allowed us to detect without ambiguity any LOH case.

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

PubMed

Pine, Sharon R; Yin, Changhong; Matloub, Yousif H; Sabaawy, Hatem E; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M Fevzi; Jayabose, Somasundaram

2005-02-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Detection of Central Nervous System Leukemia in Children with Acute Lymphoblastic Leukemia by Real-Time Polymerase Chain Reaction

PubMed Central

Pine, Sharon R.; Yin, Changhong; Matloub, Yousif H.; Sabaawy, Hatem E.; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M. Fevzi; Jayabose, Somasundaram

2005-01-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification. PMID:15681484

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas.

PubMed

Jiang, Zhiquan; Gui, Songbo; Zhang, Yazhuo

2010-09-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors.

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas

PubMed Central

JIANG, ZHIQUAN; GUI, SONGBO; ZHANG, YAZHUO

2010-01-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors. PMID:22993617

Mechanism of Glucose Regulates the Fruiting Body Formation in the Beech Culinary-Medicinal Mushroom, Hypsizygus marmoreus (Agaricomycetes).

PubMed

Zhang, Jin-Jing; Chen, Hui; Xie, Min-Ying; Chen, Ming-Jie; Hao, Hai-Bo; Wang, Hong; Feng, Zhi-Yong

2017-01-01

To understand the fruiting process of Hypsizygus marmoreus, a synthetic liquid medium (SLM) was optimized to induce fruiting body initiation. Dependent on the SLM, the effect of a monofactor (glucose) on the fruiting bodies of H. marmoreus was studied at different concentrations (10 and 40 g/L). Primordia appeared approximately 10 days earlier in low-glucose media (LGM) than in high-glucose media (HGM), whereas mature fruiting bodies formed on mushrooms approximately 7 days earlier and more primordia developed into mature fruiting bodies when cultured in HGM. In addition, the morphogenesis of the primordia was clustered in HGM, which was different than what was observed in LGM. Furthermore, differentially expressed genes (DEGs) that encoded various proteins involved in cell structure, general metabolism, signal transduction, and transcription and translation were analyzed by transcriptome sequencing. Six DEGs were detected by quantitative reverse-transcriptase polymerase chain reaction, and the results were consistent with the altered patterns of gene expression revealed by the transcriptome. This study not only identifies new candidate genes involved in the development of H. marmoreus but also provides a new research platform for studying the development of other edible mushrooms.

Loss-of-Function Mutations in a Human Gene Related to Chlamydomonas reinhardtii Dynein IC78 Result in Primary Ciliary Dyskinesia

PubMed Central

Pennarun, Gaëlle; Escudier, Estelle; Chapelin, Catherine; Bridoux, Anne-Marie; Cacheux, Valère; Roger, Gilles; Clément, Annick; Goossens, Michel; Amselem, Serge; Duriez, Bénédicte

1999-01-01

Summary Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects. PMID:10577904

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms.

PubMed

Iacoangeli, Anna; Lui, Anita; Haines, Ashley; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2017-09-01

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ-expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM + cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ + splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ + cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ. Copyright © 2017 by The American Association of Immunologists, Inc.

Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network

PubMed Central

Zhang, Zhe; Tsukikawa, Mai; Peng, Min; Polyak, Erzsebet; Nakamaru-Ogiso, Eiko; Ostrovsky, Julian; McCormack, Shana; Place, Emily; Clarke, Colleen; Reiner, Gail; McCormick, Elizabeth; Rappaport, Eric; Haas, Richard; Baur, Joseph A.; Falk, Marni J.

2013-01-01

Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. Mechanism(s) by which RC dysfunction causes global cellular sequelae are poorly understood. To identify a common cellular response to RC disease, integrated gene, pathway, and systems biology analyses were performed in human primary RC disease skeletal muscle and fibroblast transcriptomes. Significant changes were evident in muscle across diverse RC complex and genetic etiologies that were consistent with prior reports in other primary RC disease models and involved dysregulation of genes involved in RNA processing, protein translation, transport, and degradation, and muscle structure. Global transcriptional and post-transcriptional dysregulation was also found to occur in a highly tissue-specific fashion. In particular, RC disease muscle had decreased transcription of cytosolic ribosomal proteins suggestive of reduced anabolic processes, increased transcription of mitochondrial ribosomal proteins, shorter 5′-UTRs that likely improve translational efficiency, and stabilization of 3′-UTRs containing AU-rich elements. RC disease fibroblasts showed a strikingly similar pattern of global transcriptome dysregulation in a reverse direction. In parallel with these transcriptional effects, RC disease dysregulated the integrated nutrient-sensing signaling network involving FOXO, PPAR, sirtuins, AMPK, and mTORC1, which collectively sense nutrient availability and regulate cellular growth. Altered activities of central nodes in the nutrient-sensing signaling network were validated by phosphokinase immunoblot analysis in RC inhibited cells. Remarkably, treating RC mutant fibroblasts with nicotinic acid to enhance sirtuin and PPAR activity also normalized mTORC1 and AMPK signaling, restored NADH/NAD+ redox balance, and improved cellular respiratory capacity. These data specifically highlight a common pathogenesis extending across different molecular and biochemical etiologies of individual RC disorders that involves global transcriptome modifications. We further identify the integrated nutrient-sensing signaling network as a common cellular response that mediates, and may be amenable to targeted therapies for, tissue-specific sequelae of primary mitochondrial RC disease. PMID:23894440

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

PubMed

Bergmann, D J; Arciero, D M; Hooper, A B

1994-06-01

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

A recessive contiguous gene deletion of chromosome 2p16 associated with cystinuria and a mitochondrial disease.

PubMed

Parvari, R; Brodyansky, I; Elpeleg, O; Moses, S; Landau, D; Hershkovitz, E

2001-10-01

Deletions ranging from 100 Kb to 1 Mb--too small to be detected under the microscope--may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cbeta gene (PP2Cbeta), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes' products, one of which may be essential for the synthesis of mitochondrial encoded proteins.

Rearrangement of Immunoglobulin Genes in Shark Germ Cells

PubMed Central

Lee, Susan S.; Fitch, David; Flajnik, Martin F.; Hsu, Ellen

2000-01-01

The variable (V), (diversity [D]), and joining (J) region recombinases (recombination activating genes [RAGs]) can perform like transposases and are thought to have initiated development of the adaptive immune system in early vertebrates by splitting archaic V genes with transposable elements. In cartilaginous fishes, the immunoglobulin (Ig) light chain genes are organized as multiple VJ-constant (C) clusters; some loci are capable of rearrangement while others contain fused VJ. The latter may be key to understanding the evolutionary role of RAG. Are they relics of the archaic genes, or are they results of rearrangement in germ cells? Our data suggest that some fused VJ genes are not only recently rearranged, but also resulted from RAG-like activity involving hairpin intermediates. Expression studies show that these, like some other germline-joined Ig sequences, are expressed at significant levels only early in ontogeny. We suggest that a rejoined Ig gene may not merely be a sequence restricting antibody diversity, but is potentially a novel receptor no longer tied to somatic RAG expression and rearrangement. From the combined data, we arrived at the unexpected conclusion that, in some vertebrates, RAG is still an active force in changing the genome. PMID:10811858

KMeyeDB: a graphical database of mutations in genes that cause eye diseases.

PubMed

Kawamura, Takashi; Ohtsubo, Masafumi; Mitsuyama, Susumu; Ohno-Nakamura, Saho; Shimizu, Nobuyoshi; Minoshima, Shinsei

2010-06-01

KMeyeDB (http://mutview.dmb.med.keio.ac.jp/) is a database of human gene mutations that cause eye diseases. We have substantially enriched the amount of data in the database, which now contains information about the mutations of 167 human genes causing eye-related diseases including retinitis pigmentosa, cone-rod dystrophy, night blindness, Oguchi disease, Stargardt disease, macular degeneration, Leber congenital amaurosis, corneal dystrophy, cataract, glaucoma, retinoblastoma, Bardet-Biedl syndrome, and Usher syndrome. KMeyeDB is operated using the database software MutationView, which deals with various characters of mutations, gene structure, protein functional domains, and polymerase chain reaction (PCR) primers, as well as clinical data for each case. Users can access the database using an ordinary Internet browser with smooth user-interface, without user registration. The results are displayed on the graphical windows together with statistical calculations. All mutations and associated data have been collected from published articles. Careful data analysis with KMeyeDB revealed many interesting features regarding the mutations in 167 genes that cause 326 different types of eye diseases. Some genes are involved in multiple types of eye diseases, whereas several eye diseases are caused by different mutations in one gene.

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

PubMed Central

Goodhardt, M; Babinet, C; Lutfalla, G; Kallenbach, S; Cavelier, P; Rougeon, F

1989-01-01

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo. Images PMID:2508061

Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

PubMed

Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

2017-10-01

Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene expression, in those cells. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Use of gene expression data to determine effects on gonad phenotype in Japanese medaka after exposure to trenbolone or estradiol.

PubMed

Flynn, Kevin; Swintek, Joe; Johnson, Rodney

2013-06-01

Various aquatic bioassays using one of several fish species have been developed or are in the process of being developed by organizations like the US Environmental Protection Agency and the Office of Economic Cooperation and Development for testing potential endocrine-disrupting chemicals (EDCs). Often, these involve assessment of the gonad phenotype of individuals as a key endpoint that is inputted into a risk or hazard assessment. Typically, gonad phenotype is determined histologically, which involves specialized and time-consuming techniques. The methods detailed here utilize an entirely different methodology, reverse-transcription quantitative polymerase chain reaction, to determine the relative expression levels of 4 genes after exposure to either 17β-estradiol or 17β-trenbolone and, by extension, the effects of EDCs on the phenotypic status of the gonad. The 4 genes quantified, Sox9b, protamine, Fig1α, and ZPC1, are all involved in gonad development and maintenance in Japanese medaka (Oryzias latipes); these data were then inputted into a permutational multivariate analysis of variance to determine whether significant differences exist between treatment groups. This information in conjunction with the sexual genotype, which can be determined in medaka, can be used to determine adverse effects of exposure to EDCs in a similar fashion to the histologically determined gonad phenotype. Copyright © 2013 SETAC.

Biodegradability of HCH in agricultural soils from Guadeloupe (French West Indies): identification of the lin genes involved in the HCH degradation pathway.

PubMed

Laquitaine, L; Durimel, A; de Alencastro, L F; Jean-Marius, C; Gros, O; Gaspard, S

2016-01-01

Banana has been a main agricultural product in the French West Indies (Guadeloupe and Martinique) since the 1960s. This crop requires the intensive use of pesticides to prevent attacks by insect pests. Chlorinated pesticides, such as hexachlorocyclohexane (HCH), chlordecone and dieldrin, were used until the beginning of the 1990s, resulting in a generalized diffuse contamination of the soil and water in the areas of banana production, hence the need to develop solutions for cleanup of the polluted sites. The aims of this work were (i) to assess lindane degradation in soil slurry microcosms treated with lindane at 10 mg/L and (ii) to detect the catabolic genes involved in the HCH degradation pathway. The soil slurry microcosm system showed a 40% lindane degradation efficiency at the end of a 30-day experiment. Lower lindane removal was also detected in the abiotic controls, probably caused by pesticide adsorption to soil particles. Indeed, the lindane concentration decreased from 6000 to 1330 ng/mL and from 800 to 340 ng/mL for the biotic and abiotic soils, respectively. Nevertheless, some of the genes involved in the HCH degradation pathway were amplified by polymerase chain reaction (PCR) from crude deoxyribonucleic acid (DNA) extracted from the Guadeloupe agricultural soil, suggesting that HCH degradation is probably mediated by bacteria closely related to the family Sphingomonadaceae.

Medium Chain Triglycerides enhances exercise endurance through the increased mitochondrial biogenesis and metabolism.

PubMed

Wang, Ying; Liu, Zhenzhen; Han, Yi; Xu, Jiping; Huang, Wen; Li, Zhaoshen

2018-01-01

Medium Chain Triglycerides (MCT) is a dietary supplement and usually used along with medications for treating food absorption disorders including diarrhea, steatorrhea and liver disease. It has been shown that MCT plays a role in lowering weight, and decreasing metabolic syndrome, abdominal obesity and inflammation. However, it is still unknown whether MCT enhances exercise endurance. Here, we demonstrated that MCT containing diet improves high temperature induced exercise performance impairment. We found that MCT up-regulates the expression and protein levels of genes involved in mitochondrial biogenesis and metabolism. Further investigation demonstrated that the increased mitochondrial biogenesis and metabolism is mediated through the activation of Akt and AMPK signaling pathways and inhibition of TGF-β signaling pathway. Collectively, our findings indicate a beneficial effect of dietary MCT in exercise performance through the increase of mitochondrial biogenesis and metabolism.

ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use.

PubMed

Wang, Mingxuan; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Wei; Chen, Yong Q

2013-12-01

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis.

PubMed

Gilbert, Stephanie; Hood, LaChae; Seah, Stephen Y K

2018-01-15

The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth ( igr ) operon of Mycobacterium tuberculosis , catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of chsH2 , i.e., ltp2 , was expressed in recombinant Rhodococcus jostii RHA1 in combination with other genes within the igr operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a K m of 6.54 ± 0.90 μM and a k cat of 159 ± 8.50 s -1 ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria. IMPORTANCE The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth ( igr ) operon of M. tuberculosis , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice. Copyright © 2017 American Society for Microbiology.

Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse.

PubMed

Tallmadge, Rebecca L; Tseng, Chia T; Felippe, M Julia B

2014-10-01

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire. Copyright © 2014 Elsevier Ltd. All rights reserved.

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp.

PubMed

Van Hellemond, J J; Simons, B; Millenaar, F F; Tielens, A G

1998-01-01

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.

Simplification of Markov chains with infinite state space and the mathematical theory of random gene expression bursts.

PubMed

Jia, Chen

2017-09-01

Here we develop an effective approach to simplify two-time-scale Markov chains with infinite state spaces by removal of states with fast leaving rates, which improves the simplification method of finite Markov chains. We introduce the concept of fast transition paths and show that the effective transitions of the reduced chain can be represented as the superposition of the direct transitions and the indirect transitions via all the fast transition paths. Furthermore, we apply our simplification approach to the standard Markov model of single-cell stochastic gene expression and provide a mathematical theory of random gene expression bursts. We give the precise mathematical conditions for the bursting kinetics of both mRNAs and proteins. It turns out that random bursts exactly correspond to the fast transition paths of the Markov model. This helps us gain a better understanding of the physics behind the bursting kinetics as an emergent behavior from the fundamental multiscale biochemical reaction kinetics of stochastic gene expression.

Simplification of Markov chains with infinite state space and the mathematical theory of random gene expression bursts

NASA Astrophysics Data System (ADS)

Jia, Chen

2017-09-01

Here we develop an effective approach to simplify two-time-scale Markov chains with infinite state spaces by removal of states with fast leaving rates, which improves the simplification method of finite Markov chains. We introduce the concept of fast transition paths and show that the effective transitions of the reduced chain can be represented as the superposition of the direct transitions and the indirect transitions via all the fast transition paths. Furthermore, we apply our simplification approach to the standard Markov model of single-cell stochastic gene expression and provide a mathematical theory of random gene expression bursts. We give the precise mathematical conditions for the bursting kinetics of both mRNAs and proteins. It turns out that random bursts exactly correspond to the fast transition paths of the Markov model. This helps us gain a better understanding of the physics behind the bursting kinetics as an emergent behavior from the fundamental multiscale biochemical reaction kinetics of stochastic gene expression.

Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

PubMed

Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

2011-09-01

A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

Processing of alkylcobalamins in mammalian cells: a role for the MMACHC (cblC) gene product

PubMed Central

Hannibal, Luciana; Kim, Jihoe; Brasch, Nicola E.; Wang, Sihe; Rosenblatt, David S.; Banerjee, Ruma; Jacobsen, Donald W.

2009-01-01

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B12). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [57Co]-AdoCbl and MeCbl analogs consisting of [57Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [57Co]-AdoCbl was converted to [57Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5′-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from 3 patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [57Co]-propylcobalamin to [57Co]-AdoCbl and [57Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells. PMID:19447654

Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product.

PubMed

Hannibal, Luciana; Kim, Jihoe; Brasch, Nicola E; Wang, Sihe; Rosenblatt, David S; Banerjee, Ruma; Jacobsen, Donald W

2009-08-01

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B(12)). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [(57)Co]-AdoCbl and MeCbl analogs consisting of [(57)Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [(57)Co]-AdoCbl was converted to [(57)Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5'-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [(57)Co]-propylcobalamin to [(57)Co]-AdoCbl and [(57)Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.

Genomic insights into the uncultured genus 'Candidatus Magnetobacterium' in the phylum Nitrospirae.

PubMed

Lin, Wei; Deng, Aihua; Wang, Zhang; Li, Ying; Wen, Tingyi; Wu, Long-Fei; Wu, Martin; Pan, Yongxin

2014-12-01

Magnetotactic bacteria (MTB) of the genus 'Candidatus Magnetobacterium' in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magnetosomes in multiple bundles of chains per cell. These bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and sulfur. However, except for a few short genomic fragments, no genome data are available for this ecologically important genus, and little is known about their metabolic capacity owing to the lack of pure cultures. Here we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named 'Ca. Magnetobacterium casensis' isolated from Lake Miyun, China. The genome sequence indicates an autotrophic lifestyle using the Wood-Ljungdahl pathway for CO2 fixation, which has not been described in any previously known MTB or Nitrospirae organisms. Pathways involved in the denitrification, sulfur oxidation and sulfate reduction have been predicted, indicating its considerable capacity for adaptation to variable geochemical conditions and roles in local biogeochemical cycles. Moreover, we have identified a complete magnetosome gene island containing mam, mad and a set of novel genes (named as man genes) putatively responsible for the formation of bullet-shaped magnetite magnetosomes and the arrangement of multiple magnetosome chains. This first comprehensive genomic analysis sheds light on the physiology, ecology and biomineralization of the poorly understood 'Ca. Magnetobacterium' genus.

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy.

PubMed

Hettinger, A M; Allen, M R; Zhang, B R; Goad, D W; Malayer, J R; Geisert, R D

2001-08-01

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.

Differentially expressed genes of Coptotermes formosanus (Isoptera: Rhinotermitidae) challenged by chemical insecticides.

PubMed

Zhang, Yi; Zhao, Yuanyuan; Qiu, Xuehong; Han, Richou

2013-08-01

Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) termites are harmful social insects to wood constructions. The current control methods heavily depend on the chemical insecticides with increasing resistance. Analysis of the differentially expressed genes mediated by chemical insecticides will contribute to the understanding of the termite resistance to chemicals and to the establishment of alternative control measures. In the present article, a full-length cDNA library was constructed from the termites induced by a mixture of commonly used insecticides (0.01% sulfluramid and 0.01% triflumuron) for 24 h, by using the RNA ligase-mediated Rapid Amplification cDNA End method. Fifty-eight differentially expressed clones were obtained by polymerase chain reaction and confirmed by dot-blot hybridization. Forty-six known sequences were obtained, which clustered into 33 unique sequences grouped in 6 contigs and 27 singlets. Sixty-seven percent (22) of the sequences had counterpart genes from other organisms, whereas 33% (11) were undescribed. A Gene Ontology analysis classified 33 unique sequences into different functional categories. In general, most of the differential expression genes were involved in binding and catalytic activity.

[Transmission disequilibrium test for nonsyndromic cleft lip and palate and segment homeobox gene-1 gene].

PubMed

Wu, Ping-An; Li, Yun-Liang; Wu, Han-Jiang; Wang, Kai; Fan, Guo-Zheng

2007-09-01

To investigate the relationship between muscle segment homeobox gene-1 (MSX1) and the genetic susceptibility of nonsyndromic cleft lip and palate (NSCLP) in Hunan Hans. One microsatellite DNA marker CA repeat in MSX1 intron region was used as genetic marker. The genotypes of 387 members in 129 NSCLP nuclear family trios were analyzed by polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis. Then transmission disequilibrium test (TDT) and Logistic regression analysis were used to conduct association analysis. TDT analysis confirmed that CA4 allele in CL/P and CPO groups preferentially transmitted to the affected offspring (P = 0.018, P = 0.041). Logistic regression analysis indicated that the recessive model of inheritance was supported, and CA4 itself or CA4 acting as a marker for a disease allele or haplotype was inherited in a recessive fashion (P = 0.009). MSX1 gene is associated with NSCLP, and MSX1 gene may be directly involved either in the etiology of NSCLP or in linkage disequilibrium with disease-predisposing sites.

Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening.

PubMed

Liu, Juhua; Xu, Biyu; Hu, Lifang; Li, Meiying; Su, Wei; Wu, Jing; Yang, Jinghao; Jin, Zhiqiang

2009-01-01

To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene.

Meta-analysis of studies using suppression subtractive hybridization and microarrays to investigate the effects of environmental stress on gene transcription in oysters.

PubMed

Anderson, Kelli; Taylor, Daisy A; Thompson, Emma L; Melwani, Aroon R; Nair, Sham V; Raftos, David A

2015-01-01

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

PubMed

Ciupescu, Laurentiu-Mihai; Auvray, Frederic; Nicorescu, Isabela Madalina; Meheut, Thomas; Ciupescu, Veronica; Lardeux, Anne-Laure; Tanasuica, Rodica; Hennekinne, Jacques-Antoine

2018-06-05

To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis (PFGE) techniques. Nineteen CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene (se). The Calarași outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Pitești outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

Possible roles for products of polymorphic MHC and linked olfactory receptor genes during selection processes in reproduction.

PubMed

Ziegler, Andreas; Dohr, Gotrfried; Uchanska-Ziegler, Barbara

2002-07-01

Polymorphic genes of the human major histocompatibility complex [MHC; human leukocyte antigen (HLA)] are probably important in determining resistance to parasites and avoidance of inbreeding. We investigated whether HLA-associated sexual selection could also involve HLA-linked olfactory receptor (OR) genes, which might not only participate in olfaction-guided mate choice, but also in selection processes within the testis. The testicular expression status of HLA class I molecules (by immunohistology) and HLA-linked OR genes (by transcriptional analysis) was determined. Various HLA class I heavy chains, but not beta2-microglobulin (beta2m), were expressed, mainly at the spermatocyte I stage. Of 17 HLA-linked OR genes analyzed, eight were found to be transcribed in the testis. They exhibited varying numbers of 5'- or 3'-non-coding exons as well as differential splicing. We suggest that testis-expressed polymorphic HLA and OR proteins are functionally connected and serve the selection of spermatozoa, enabling them to distinguish 'self from 'non-self [the sperm-receptor-selection (SRS) hypothesis].

Meta-Analysis of Studies Using Suppression Subtractive Hybridization and Microarrays to Investigate the Effects of Environmental Stress on Gene Transcription in Oysters

PubMed Central

Thompson, Emma L.; Melwani, Aroon R.; Nair, Sham V.; Raftos, David A.

2015-01-01

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters. PMID:25768438

Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.)

PubMed Central

Iino, Shiori; Kubo, Takeo

2018-01-01

ABSTRACT Although the molecular mechanisms involved in learning and memory in insects have been studied intensively, the intracellular signaling mechanisms involved in early memory formation are not fully understood. We previously demonstrated that phospholipase C epsilon (PLCe), whose product is involved in calcium signaling, is almost selectively expressed in the mushroom bodies, a brain structure important for learning and memory in the honeybee. Here, we pharmacologically examined the role of phospholipase C (PLC) in learning and memory in the honeybee. First, we identified four genes for PLC subtypes in the honeybee genome database. Quantitative reverse transcription-polymerase chain reaction revealed that, among these four genes, three, including PLCe, were expressed higher in the brain than in sensory organs in worker honeybees, suggesting their main roles in the brain. Edelfosine and neomycin, pan-PLC inhibitors, significantly decreased PLC activities in homogenates of the brain tissues. These drugs injected into the head of foragers significantly attenuated memory acquisition in comparison with the control groups, whereas memory retention was not affected. These findings suggest that PLC in the brain is involved in early memory formation in the honeybee. To our knowledge, this is the first report of a role for PLC in learning and memory in an insect. PMID:29330349

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

PubMed Central

Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P

2000-01-01

Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670

Plastidial Disproportionating Enzyme Participates in Starch Synthesis in Rice Endosperm by Transferring Maltooligosyl Groups from Amylose and Amylopectin to Amylopectin1

PubMed Central

Dong, Xiangbai; Zhang, Du; Liu, Jie; Liu, Hualiang; Tian, Lihong; Jiang, Ling

2015-01-01

Plastidial disproportionating enzyme1 (DPE1), an α-1,4-d-glucanotransferase, has been thought to be involved in storage starch synthesis in cereal crops. However, the precise function of DPE1 remains to be established. We present here the functional identification of DPE1 in storage starch synthesis in rice (Oryza sativa) by endosperm-specific gene overexpression and suppression. DPE1 overexpression decreased amylose content and resulted in small and tightly packed starch granules, whereas DPE1 suppression increased amylose content and formed heterogeneous-sized, spherical, and loosely packed starch granules. Chains with degree of polymerization (DP) of 6 to 10 and 23 to 38 were increased, while chains with DP of 11 to 22 were decreased in amylopectin from DPE1-overexpressing seeds. By contrast, chains with DP of 6 to 8 and 16 to 36 were decreased, while chains with DP of 9 to 15 were increased in amylopectin from DPE1-suppressed seeds. Changes in DPE1 gene expression also resulted in modifications in the thermal and pasting features of endosperm starch granules. In vitro analyses revealed that recombinant DPE1 can break down amylose into maltooligosaccharides in the presence of Glc, while it can transfer maltooligosyl groups from maltooligosaccharide to amylopectin or transfer maltooligosyl groups within and among amylopectin molecules in the absence of Glc. Moreover, a metabolic flow of maltooligosyl groups from amylose to amylopectin was clearly identifiable when comparing DPE1-overexpressing lines with DPE1-suppressed lines. These findings demonstrate that DPE1 participates substantially in starch synthesis in rice endosperm by transferring maltooligosyl groups from amylose and amylopectin to amylopectin. PMID:26471894

Differentially expressed genes in nonsmall cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer.

PubMed

Kettunen, Eeva; Anttila, Sisko; Seppänen, Jouni K; Karjalainen, Antti; Edgren, Henrik; Lindström, Irmeli; Salovaara, Reijo; Nissén, Anna-Maria; Salo, Jarmo; Mattson, Karin; Hollmén, Jaakko; Knuutila, Sakari; Wikman, Harriet

2004-03-01

The expression patterns of cancer-related genes in 13 cases of squamous cell lung cancer (SCC) were characterized and compared with those in normal lung tissue and 13 adenocarcinomas (AC), the other major type of nonsmall cell lung cancer (NSCLC). cDNA array was used to screen the gene expression levels and the array results were verified using a real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Thirty-nine percent of the 25 most upregulated and the 25 most downregulated genes were common to SCC and AC. Of these genes, DSP, HMGA1 (alias HMGIY), TIMP1, MIF, CCNB1, TN, MMP11, and MMP12 were upregulated and COPEB (alias CPBP), TYROBP, BENE, BMPR2, SOCS3, TIMP3, CAV1, and CAV2 were downregulated. The expression levels of several genes from distinct protein families (cytokeratins and hemidesmosomal proteins) were markedly increased in SCC compared with AC and normal lung. In addition, several genes, overexpressed in SCC, such as HMGA1, CDK4, IGFBP3, MMP9, MMP11, MMP12, and MMP14, fell into distinct chromosomal loci, which we have detected as gained regions on the basis of comparative genomic hybridization data. Our study revealed new candidate genes involved in NSCLC.

Identification of the Key Genes and Pathways in Esophageal Carcinoma.

PubMed

Su, Peng; Wen, Shiwang; Zhang, Yuefeng; Li, Yong; Xu, Yanzhao; Zhu, Yonggang; Lv, Huilai; Zhang, Fan; Wang, Mingbo; Tian, Ziqiang

2016-01-01

Objective . Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods . 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results . A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion . The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

Image-guided genomic analysis of tissue response to laser-induced thermal stress

NASA Astrophysics Data System (ADS)

Mackanos, Mark A.; Helms, Mike; Kalish, Flora; Contag, Christopher H.

2011-05-01

The cytoprotective response to thermal injury is characterized by transcriptional activation of ``heat shock proteins'' (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition.

PubMed

Ibrahim, Sherrine A; Ackerman, William E; Summerfield, Taryn L; Lockwood, Charles J; Schatz, Frederick; Kniss, Douglas A

2016-02-01

Inflammation is a proximate mediator of preterm birth and fetal injury. During inflammation several microRNAs (22 nucleotide noncoding ribonucleic acid (RNA) molecules) are up-regulated in response to cytokines such as interleukin-1β. MicroRNAs, in most cases, fine-tune gene expression, including both up-regulation and down-regulation of their target genes. However, the role of pro- and antiinflammatory microRNAs in this process is poorly understood. The principal goal of the work was to examine the inflammatory genomic profile of human decidual cells challenged with a proinflammatory cytokine known to be present in the setting of preterm parturition. We determined the coding (messenger RNA) and noncoding (microRNA) sequences to construct a network of interacting genes during inflammation using an in vitro model of decidual stromal cells. The effects of interleukin-1β exposure on mature microRNA expression were tested in human decidual cell cultures using the multiplexed NanoString platform, whereas the global inflammatory transcriptional response was measured using oligonucleotide microarrays. Differential expression of select transcripts was confirmed by quantitative real time-polymerase chain reaction. Bioinformatics tools were used to infer transcription factor activation and regulatory interactions. Interleukin-1β elicited up- and down-regulation of 350 and 78 nonredundant transcripts (false discovery rate < 0.1), respectively, including induction of numerous cytokines, chemokines, and other inflammatory mediators. Whereas this transcriptional response included marked changes in several microRNA gene loci, the pool of fully processed, mature microRNA was comparatively stable following a cytokine challenge. Of a total of 6 mature microRNAs identified as being differentially expressed by NanoString profiling, 2 (miR-146a and miR-155) were validated by quantitative real time-polymerase chain reaction. Using complementary bioinformatics approaches, activation of several inflammatory transcription factors could be inferred downstream of interleukin-1β based on the overall transcriptional response. Further analysis revealed that miR-146a and miR-155 both target genes involved in inflammatory signaling, including Toll-like receptor and mitogen-activated protein kinase pathways. Stimulation of decidual cells with interleukin-1β alters the expression of microRNAs that function to temper proinflammatory signaling. In this setting, some microRNAs may be involved in tissue-level inflammation during the bulk of gestation and assist in pregnancy maintenance. Copyright © 2016 Elsevier Inc. All rights reserved.

The PDI genes of wheat and their syntenic relationship to the esp2 locus of rice.

PubMed

Johnson, Joshua C; Appels, Rudi; Bhave, Mrinal

2006-04-01

The storage protein polymers in the endosperm, stabilised by disulphide bonds, determine a number of processing qualities of wheat dough. The enzyme protein disulphide isomerase (PDI), involved in the formation of disulphide bonds, is strongly suggested to play a role in the formation of wheat storage protein bodies. Reports of the rice mutant esp2 exhibiting aberrant storage protein deposition in conjunction with a lack of PDI expression provided strong indications of a direct role for PDI in storage protein deposition. The potential significance of wheat PDI prompted the present studies into exploring any orthology between wheat PDI genes and rice PDI and esp2 loci. By designing allele-specific (AS)-polymerase chain reaction (PCR) markers, two of the three wheat PDI genes could be genetically mapped to group 4 chromosomes and showed close association with GERMIN genes. Physical mapping led to localisation of wheat PDI genes to chromosomal "bins" on the proximal section of chromosome 4AL and distal sections of 4BS and 4DS. Identification of the putative PDI gene of rice and its comparison to the esp2 locus revealed that they were present at similar positions on the short arm of chromosome 11. Analysis of a large section of the PDI-containing section of rice chromosome 11S revealed a number of putative orthologues from The Institute for Genomic Research Triticum aestivum Gene Index database, of which five had been mapped, each localising to group 4 chromosomes, many in good agreement with our mapping results. The results strongly suggest a close linkage between the esp2 marker and the PDI gene of rice and an orthology between the PDI loci of rice and wheat and predict quantitative-trait loci involved in storage protein deposition at the PDI loci.

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses

PubMed Central

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants. PMID:27749935

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

PubMed

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

A novel role of the NRF2 transcription factor in the regulation of arsenite-mediated keratin 16 gene expression in human keratinocytes.

PubMed

Endo, Hitoshi; Sugioka, Yoshihiko; Nakagi, Yoshihiko; Saijo, Yasuaki; Yoshida, Takahiko

2008-07-01

Inorganic sodium arsenite (iAs) is a ubiquitous environmental contaminant and is associated with an increased risk of skin hyperkeratosis and cancer. We investigated the molecular mechanisms underlying the regulation of the keratin 16 (K16) gene by iAs in the human keratinocyte cell line HaCaT. We performed reverse transcriptase polymerase chain reaction, luciferase assays, Western blots, and electrophoretic mobility shift assays to determine the transcriptional regulation of the K16 gene by iAs. We used gene overexpression approaches to elucidate the nuclear factor erythroid-derived 2 related factor 2 (NRF2) involved in the K16 induction. iAs induced the mRNA and protein expression of K16. We also found that the expression of K16 was transcriptionally induced by iAs through activator protein-1-like sites and an antioxidant response element (ARE) in its gene promoter region. Treatment with iAs also enhanced the production and translocation of the NRF2 transcription factor, an ARE-binding protein, into the nucleus without modification of its mRNA expression. In addition, iAs elongated the half-life of the NRF2 protein. When overexpressed in HaCaT cells, NRF2 was also directly involved in not only the up-regulation of the detoxification gene thioredoxin but also K16 gene expression. Our data clearly indicate that the K16 gene is a novel target of NRF2. Furthermore, our findings also suggest that NRF2 has opposing roles in the cell--in the activation of detoxification pathways and in promoting the development of skin disorders.

Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus

PubMed Central

Holmes, Dawn E; Risso, Carla; Smith, Jessica A; Lovley, Derek R

2012-01-01

Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (>80° C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as having a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA (coenzyme A) reductase (BCR) found in all strictly anaerobic bacteria, but has instead genes coding for a bzd-type ATP-consuming class I BCR, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for phenol carboxylation proceeding through a phenylphosphate intermediate were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative reverse transcriptase polymerase chain reaction demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates vs growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism are highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets. PMID:21776029

Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells

PubMed Central

Choi, Y; Lim, SY; Jeong, HS; Koo, KA; Sung, SH; Kim, YC

2009-01-01

Background and purpose: We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45α, gadd45γ and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA. PMID:19466985

Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila.

PubMed

Thimgan, Matthew S; Seugnet, Laurent; Turk, John; Shaw, Paul J

2015-05-01

Flies mutant for the canonical clock protein cycle (cyc(01)) exhibit a sleep rebound that is ∼10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc(01) mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, we hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking. We used partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc(01) mutants that had been sleep deprived or starved for 7 h. We then used genetics to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation. Laboratory. Drosophila melanogaster. Sleep deprivation and starvation. We identified 84 genes with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc(01) mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on our data we have renamed heimdall (hll). Using lipidomics we confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls. We have identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis. © 2015 Associated Professional Sleep Societies, LLC.

Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

PubMed

Pérez-Rico, A; Crespo, F; Sanmartín, M L; De Santiago, A; Vega-Pla, J L

2014-10-01

Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B. Copyright © 2014 Elsevier B.V. All rights reserved.

Deletion patterns of the STS gene and flanking sequences in Israeli X-linked ichthyosis patients and carriers: analysis by polymerase chain reaction and fluorescence in situ hybridization techniques.

PubMed

Aviram-Goldring, A; Goldman, B; Netanelov-Shapira, I; Chen-Shtoyerman, R; Zvulunov, A; Tal, O; Ilan, T; Peleg, L

2000-03-01

Deletion of the entire steroid sulfatase (STS) gene is the most common molecular defect in X-linked ichthyosis (XLI) patients. Usually, additional flanking sequences are also missing. The aim of this study was to estimate the extent of deletions in an ethnically heterogeneous population of Israeli XLI patients. Multiplex polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques were applied in the analysis of blood samples of 24 patients and amniotic cells of seven affected fetuses from 22 unrelated families. In 19 families, a large deletion of the 2-3 megabase was found. It included the whole STS gene and spanned adjacent areas up- and downstream between the loci DXS 1139 and DXS 1132. Two unrelated families of Iraqi ancestry had a partial deletion of the gene and its centromeric adjacent sequence. In another family, the telomeric end of the extragenic segment was only partially missing. Application of FISH on metaphase blood cells and interphase amniotic cells confirmed the diagnosis of XLI in all patients, except the three with partial intragenic deletion. In those cases, the remaining fraction of the gene was sufficient to provide a false negative result. Diagnosis of carriers and prenatal diagnosis in uncultured cells was applicable only by FISH. Our study revealed a remarkable heterogeneity in the deletion pattern among Israeli patients with XLI. This heterogeneity could not be attributed to specific ethnic groups because of the small size of the study group. More studies involving patients of various ancestries should be carried out. In addition, this study demonstrated the usefulness of the FISH technique in the prenatal diagnosis of fetuses with suspected XLI.

Cold temperature blocks thyroid hormone-induced changes in lipid and energy metabolism in the liver of Lithobates catesbeianus tadpoles.

PubMed

Suzuki, Shunsuke; Awai, Koichiro; Ishihara, Akinori; Yamauchi, Kiyoshi

2016-01-01

Exposure of the American bullfrog Lithobates catesbeianus tadpoles to low temperature affects many biological processes including lipid metabolism and the thyroid hormone (TH) signaling pathway, resulting in arrest of TH-induced metamorphosis. To clarify what molecular events occur in this phenomenon, we investigated the glycerophospholipid and fatty acid (FA) compositions, the activities of mitochondrial enzymes and the transcript levels of related genes in the liver of control (26 °C) and cold-treated (4 °C) tadpoles with or without 5 nM 3,3',5-triiodothyronine (T3). Exposure to T3 decreased the tail height and polyunsaturation of FAs in the glycerophospholipids, and increased plasma glucose levels and transcript levels of primary TH-response genes including TH receptor, and some energy metabolic (cox4, srebp1 and fas) and FA chain elongase genes (elovl3 and elovl5). However, these T3-induced responses were abolished at 4 °C. Exposure to cold temperature enhanced plasma glucose, triglyceride and free FA levels, monounsaturation of FAs, mitochondrial enzymes activities (cytochrome c oxidase and carnitine palmitoyltransferase; U/g liver), with the upregulation of the genes involved in glycogenolysis (pygl), gluconeogenesis (pck1 and g6pc2), FA β-oxidation (acadl), and cholesterol uptake and synthesis (hmgcr, srebp2 and ldlr1), glycerophospholipids synthesis (pcyt1, pcyt2, pemt, and pparg), and FA monounsaturation (scd1) and chain elongation (elovl1 and elovl2). T3 had little effect on the cold-induced changes. Our study demonstrated that exposures to T3 and cold temperature exert different effects on lipid metabolism, resulting in changes in the FA composition in glycerophospholipids, and suggests that a cold-induced signal may block TH-signaling pathway around primary TH-response genes.

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1

PubMed Central

Pan, Ling; Pasternak, David A.; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W.

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function. PMID:28350844

Effects of β-glucan extracted from Agaricus blazei on the expression of ERCC5, CASP9, and CYP1A1 genes and metabolic profile in HepG2 cells.

PubMed

da Silva, A F; Sartori, D; Macedo, F C; Ribeiro, L R; Fungaro, M H P; Mantovani, M S

2013-06-01

The polysaccharide β-glucan has biological properties that stimulate the immune system and can prevent chronic pathologies, including cancer. It has been shown to prevent damage to DNA caused by the chemical and physical agents to which humans are exposed. However, the mechanism of β-glucan remains poorly understood. The objective of the present study was to verify the protective effect of β-glucan on the expression of the genes ERCC5 (involved in excision repair of DNA damage), CASP9 (involved in apoptosis), and CYP1A1 (involved in the metabolism of xenobiotics) using real-time polymerase chain reaction and perform metabolic profile measurements on the HepG2 cells. Cells were exposed to only benzo[a]pyrene (B[a]P), β-glucan, or a combination of B[a]P with β-glucan. The results demonstrated that 50 µg/mL β-glucan significantly repressed the expression of the ERCC5 gene when compared with the untreated control cells in these conditions. No change was found in the CASP9 transcript level. However, the CYP1A1 gene expression was also induced by HepG2 cells exposed to B[a]P only or in association with β-glucan, showing its effective protector against damage caused by B[a]P, while HepG2 cells exposed to only β-glucan did not show CYP1A1 modulation. The metabolic profiles showed moderate bioenergetic metabolism with an increase in the metabolites involved in bioenergetic metabolism (alanine, glutamate, creatine and phosphocholine) in cells treated with β-glucan and to a lesser extent treated with B[a]P. Thus, these results demonstrate that the chemopreventive activity of β-glucan may modulate bioenergetic metabolism and gene expression.

Mapping of the human cone transducin {alpha} subunit (GNAT2) gene to 1p13 and mutation analysis in patients with Stargardt`s disease

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

Magovcevic, I.; Weremowicz, S.; Morton, C.C.

Transducin {alpha} subunits are members of a large family of G-proteins and play an important role in phototransduction in rod and cone photoreceptors. We report the localization of the human cone {alpha} transducin (GNAT2) gene using fluorescence in situ hybridization (FISH) on chromosome 1 in band p13. The recent assignment of a gene for Stargardt`s disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. Stargardt`s disease is characterized by degeneration in late childhood or early adulthood of the macula of the retina, a region richmore » in cones. We screened patients with Stargardt`s disease, with or without peripheral cone involvement as monitored by the full-field ERG, for mutations in this gene. We investigated 66 unrelated patients including 22 with peripheral cone dysfunction for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. One patient (034-16) was heterozygous for a silent change in exon VI, Asp238Asp (GAT to GAC). Two patients, one (035-005) with peripheral cone involvement and one (071-001) without peripheral cone involvement, were heterozygous for the missense change Val124Met (GTG to ATG) in exon IV. A subsequent screen of 96 unrelated, unaffected controls revealed one individual (N10) who was also heterozygous for the Val124Met alteration. We concluded that Asp238Asp and Val124Met are rare variants not causing Stargardt`s disease. Hence, no disease-specific mutations were found indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt`s disease.« less

RNA-Seq Expression Analysis of Enteric Neuron Cells with Rotenone Treatment and Prediction of Regulated Pathways.

PubMed

Guan, Qiang; Wang, Xijin; Jiang, Yanyan; Zhao, Lijuan; Nie, Zhiyu; Jin, Lingjing

2017-02-01

The enteric nervous system (ENS) is involved in the initiation and development of the pathological process of Parkinson's disease (PD). The effect of rotenone on the ENS may trigger the progression of PD through the central nervous system (CNS). In this study, we used RNA-sequencing (RNA-seq) analysis to examine differential expression genes (DEGs) and pathways induced by in vitro treatment of rotenone in the enteric nervous cells isolated from rats. We identified 45 up-regulated and 30 down-regulated genes. The functional categorization revealed that the DEGs were involved in the regulation of cell differentiation and development, response to various stimuli, and regulation of neurogenesis. In addition, the pathway and network analysis showed that the Mitogen Activated Protein Kinase (MAPK), Toll-like receptor, Wnt, and Ras signaling pathways were intensively involved in the effect of rotenone on the ENS. Additionally, the quantitative real-time polymerase chain reaction result for the selected seven DEGs matched those of the RNA-seq analysis. Our results present a significant step in the identification of DEGs and provide new insight into the progression of PD in the rotenone-induced model.

Three mouse models of human thalassemia.

PubMed Central

Martinell, J; Whitney, J B; Popp, R A; Russell, L B; Anderson, W F

1981-01-01

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes. Images PMID:6946454

Biased immunoglobulin light chain gene usage in the shark1

PubMed Central

Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2015-01-01

This study of a large family of kappa light (L) chain clusters in nurse shark completes the characterization of its classical immunoglobulin (Ig) gene content (two heavy chain classes, mu and omega, and four L chain isotopes, kappa, lambda, sigma, and sigma-2). The shark kappa clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over a ~500 bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ca. 39 kappa clusters are pre-rearranged in the germline (GL-joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, non-productive, and sterile transcripts of the kappa clusters compared to the other three L chain isotypes. Kappa cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and non-productive rearrangements. These results show that the individual activation of the spatially distant kappa clusters is non-random. Although both split and GL-joined kappa genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. PMID:26342033

Biased Immunoglobulin Light Chain Gene Usage in the Shark.

PubMed

Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2015-10-15

This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. Copyright © 2015 by The American Association of Immunologists, Inc.

relA-dependent RNA polymerase activity in Escherichia coli.

PubMed Central

Ryals, J; Bremer, H

1982-01-01

Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase. The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total instantaneous rate of RNA synthesis) decreased 1.7-fold in the relA+ strain and increased 1.9-fold in the relA mutant; and (iv) the RNA polymerase activity (measured by the percentage of total RNA polymerase enzyme active in transcription an any instant) decreased from 20 to 3.6% in the relA+ strain and remained unchanged (or increased at most to 22%) in the relA mutant. It is suggested that both rRNA gene activity and the RNA polymerase activity depend on the intracellular concentration of guanosine tetraphosphate, whereas the altered chain elongation rate and stability of rRNA are temperature or amino acid starvation effects, respectively, without involvement of relA function. PMID:6174501

Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain

PubMed Central

Aline-Fardin, Aude; Bender, Sebastien; Fabiani, Bettina; Buob, David; Brahimi, Said; Verpont, Marie Christine; Mothy, Mohamad; Ronco, Pierre; Boffa, Jean Jacques; Aucouturier, Pierre; Garderet, Laurent

2015-01-01

Abstract Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available. A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-20∗01/Jk2∗01 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation. The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission. CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case. PMID:26266355

Catalytic and reactive polypeptides and methods for their preparation and use

DOEpatents

Schultz, Peter

1994-01-01

Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like.

Chromosome rearrangements in canine fibrosarcomas.

PubMed

Sargan, D R; Milne, B S; Hernandez, J Aguirre; O'Brien, P C M; Ferguson-Smith, M A; Hoather, T; Dobson, J M

2005-01-01

We have previously reported the use of six- and seven-color paint sets in the analysis of canine soft tissue sarcomas. Here we combine this technique with flow sorting of translocation chromosomes, reverse painting, and polymerase chain reaction (PCR) analysis of the gene content of the reverse paint in order to provide a more detailed analysis of cytogenetic abnormalities in canine tumors. We examine two fibrosarcomas, both from female Labrador retrievers, and show abnormalities in chromosomes 11 and 30 in both cases. Evidence of involvement of TGFBR1 is presented for one tumor.

Somatic hypermutation and junctional diversification at Ig heavy chain loci in the nurse shark.

PubMed

Malecek, Karolina; Brandman, Julie; Brodsky, Jennie E; Ohta, Yuko; Flajnik, Martin F; Hsu, Ellen

2005-12-15

We estimate there are approximately 15 IgM H chain loci in the nurse shark genome and have characterized one locus. It consists of one V, two D, and one J germline gene segments, and the constant (C) region can be distinguished from all of the others by a unique combination of restriction endonuclease sites in Cmu2. On the basis of these Cmu2 markers, 22 cDNA clones were selected from an epigonal organ cDNA library from the same individual; their C region sequences proved to be the same up to the polyadenylation site. With the identification of the corresponding germline gene segments, CDR3 from shark H chain rearrangements could be analyzed precisely, for the first time. Considerable diversity was generated by trimming and N addition at the three junctions and by varied recombination patterns of the two D gene segments. The cDNA sequences originated from independent rearrangements events, and most carried both single and contiguous substitutions. The 53 point mutations occurred with a bias for transition changes (53%), whereas the 78 tandem substitutions, mostly 2-4 bp long, do not (36%). The nature of the substitution patterns is the same as for mutants from six loci of two nurse shark L chain isotypes, showing that somatic hypermutation events are very similar at both H and L chain genes in this early vertebrate. The cis-regulatory elements targeting somatic hypermutation must have already existed in the ancestral Ig gene, before H and L chain divergence.

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin*

PubMed Central

Chan, Linda J.; Rosengren, K. Johan; Layfield, Sharon L.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed A.; Wade, John D.

2012-01-01

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4–24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin. PMID:23024363

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

[Clinical and genetic characteristics of children with Leigh syndrome].

PubMed

Fang, F; Shen, Y; Shen, D M; Liu, Z M; Ding, C H; Zhang, W C; Sun, S Z; Lyu, J L; Han, T L; Wang, X H; Zhang, W H; Yang, X Y; Li, J W; Wu, H S

2017-03-02

Objective: To investigate the clinically and genetic characteristics of children with Leigh syndrome. Method: Patients with clinically diagnosed Leigh syndrome(LS)in the department of Neurology, Beijing Children's Hospital from January 2013 to February 2016 underwent the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) detecting with next generation sequencing (NGS) technology. The clinical data of gene confirmed cases were retrospectively collected and analyzed. The differences in the onset age, clinical manifestations, lactic acid level and MRI results between the mtDNA variation and nDNA variation were compared and analyzed. t test, Chi-square test and Fisher's exact test were used for statistical analysis. Result: Thirty-five cases were diagnosed by gene detection, including 20 males and 15 females. The median onset age was 1 year (ranging from the neonatal period to 4.4 years old). The age of onset within 2 years accounted for 74%(26 cases). The onset age of initial symptoms, including developmental delay, developmental regression, and seizures, were 6 (4, 12) months, 12 (8, 14) months, and 6 (1, 23) months respectively. The onset age of ptosis, extrapyramidal symptoms and ataxia were 26 (18, 44) months, 28 (23, 40) months and 28 (19, 35) months, respectively. There were significant differences in the onset age between the three groups ( H =21.919, P =0.01). Within the 35 cases, 29 were manifested with developmental delay (83%), 26 with dystonia (74%), 18 with growth retardation, 15 with myasthenia, 13 with developmental regression, 11 with dysphagia, 10 with feeding difficulties, 4 with skeletal dysplasia, and 2 with digestive tract symptoms; nystagmus and respiratory abnormalities were observed in 9 cases respectively; extrapyramidal symptoms, peripheral nerve injury, ptosis, seizures were observed in 8 cases respectively; and ataxia, ophthalmoplegia and hypertrichiasis were found in 5 cases respectively.The blood lactic acid was measured in 32 LS patients, within which 23 cases (72%) had increased results; 8 out of 11 cases who underwent were cerebrospinal fluid lactic acid test had increased results. The results of neuroimaging revealed that all the patients were involved in the brainstem and (or) basal ganglia, of whom 27 (77%) had brainstem involvement, 24 (69%) had basal ganglia involvement. Thirteen out of 14 patients who had medulla oblongata involvement had nDNA variation; while 7 out of 8 patients with cerebellar involvement had nDNA variation. Genetic etiology was confirmed in all patients, among whom there were 17 cases (49%) with mtDNA mutation, including 8993T>C/G ( n =5), 14487T>C ( n =4), 13513G>A ( n =2), 9176T>C, 10158T>C, 3697G>A, 10191T>C, 14459A>G and 11777C>A ( n =1) respectively. Remaining 18 cases(51%) had nDNA mutation, including SURF1 gene( n =10), PDHA1 gene( n =3) and one case each of NDUFV1, NDUFAF6, NDUFAF5, NDUFS1 and COQ7 genes. In this study, 27 types of mutations were founded, 15 of which had not been previously reported. Respiratory chain gene mutations have been found in 31 cases(89%); 3 cases had PDHc gene mutations, and 1 case had other mutation. Conclusion: LS usually occurs in infants. The most common primary symptoms are age-dependent abnormal movements, ocular symptoms, and seizures. Respiratory chain defects is the most common causes of LS.SURF1 is the most common variation, followed by 8993T>C/G, 14487 T>C and 13513G>A mutation.

Phylogenetic grouping and distribution of virulence genes in Escherichia coli along the production and supply chain of pork around Hubei, China.

PubMed

Khan, Sher Bahadar; Zou, Geng; Cheng, Yu-Ting; Xiao, Ran; Li, Lu; Wu, Bin; Zhou, Rui

2017-06-01

Escherichia coli is an important foodborne zoonotic pathogen. A total of 285 strains of E. coli were isolated from the production and supply chain of pork in Hubei, China and characterized. Their phylogroups (A, B1, B2, and D) and virulence genes of public health importance become more and more diverse along the production and supply chain. Copyright © 2016. Published by Elsevier B.V.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

Agaricus brasiliensis (sun mushroom) affects the expression of genes related to cholesterol homeostasis.

PubMed

de Miranda, Aline Mayrink; Rossoni Júnior, Joamyr Victor; Souza E Silva, Lorena; Dos Santos, Rinaldo Cardoso; Silva, Marcelo Eustáquio; Pedrosa, Maria Lúcia

2017-06-01

The sun mushroom (Agaricus brasiliensis) is considered a major source of bioactive compounds with potential health benefits. Mushrooms typically act as lipid-lowering agents; however, little is known about the mechanisms of action of A. brasiliensis in biological systems. This study aimed to determine the underlying mechanism involved in the cholesterol-lowering effect of A. brasiliensis through the assessment of fecal and serum lipid profiles in addition to gene expression analysis of specific transcription factors, enzymes, and transporters involved in cholesterol homeostasis. Twenty-four albino Fischer rats approximately 90 days old, with an average weight of 205 g, were divided into four groups of 6 each and fed a standard AIN-93 M diet (C), hypercholesterolemic diet (H), hypercholesterolemic diet +1 % A. brasiliensis (HAb), or hypercholesterolemic diet +0.008 % simvastatin (HS) for 6 weeks. Simvastatin was used as a positive control, as it is a typical drug prescribed for lipid disorders. Subsequently, blood, liver, and feces samples were collected for lipid profile and quantitative real-time polymerase chain reaction gene expression analyses. Diet supplementation with A. brasiliensis significantly improved serum lipid profiles, comparable to the effect observed for simvastatin. In addition, A. brasiliensis dietary supplementation markedly promoted fecal cholesterol excretion. Increased expression of 7α-hydroxylase (CYP7A1), ATP-binding cassette subfamily G-transporters (ABCG5/G8), and low-density lipoprotein receptor (LDLR) was observed following A. brasiliensis administration. Our results suggest that consumption of A. brasiliensis improves the serum lipid profile in hypercholesterolemic rats by modulating the expression of key genes involved in hepatic cholesterol metabolism.

The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR.

PubMed

Retière, C; Halary, F; Peyrat, M A; Le Deist, F; Bonneville, M; Hallet, M M

1999-01-15

Functional chimeric TCR chains, encoded by V gamma J gamma C beta or V gamma J beta C beta hybrid gene TCR, are expressed at the surface of a small fraction of alpha beta T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR gamma and beta loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between D beta and J gamma genic segments generates C gamma V beta and C beta V gamma hybrid loci. Within the C gamma V beta locus, secondary rearrangements between V gamma and J gamma or V gamma and J beta elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the C beta V gamma locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same D beta-3' recombination signal sequence can be used in successive rearrangements with J gamma and J beta genic segments, thus showing that a signal joint has been involved in a secondary recombination event.

Silk Gland Gene Expression during Larval-Pupal Transition in the Cotton Leaf Roller Sylepta derogata (Lepidoptera: Pyralidae)

PubMed Central

Su, Honghua; Cheng, Yuming; Wang, Zhongyang; Li, Zhong; Stanley, David; Yang, Yizhong

2015-01-01

The cotton leaf roller, Sylepta derogata, is a silk-producing insect pest. While young larvae feed on the underside of leaves, the older ones roll cotton leaves and feed on the leaf edges, which defoliates cotton plants. The larvae produce silk to stabilize the rolled leaf and to balloon from used to new leaves. Despite the significance of silk in the biology of pest insect species, there is virtually no information on the genes involved in their silk production. This is a substantial knowledge gap because some of these genes may be valuable targets for developing molecular pest management technologies. We addressed the gap by posing the hypothesis that silk gland gene expression changes during the transition from larvae to pupae. We tested our hypothesis using RNA-seq to investigate changes in silk gland gene expression at three developmental stages, 5th instar larvae (silk producing; 15,445,926 clean reads), prepupae (reduced silk producing; 13,758,154) and pupae (beyond silk producing; 16,787,792). We recorded 60,298 unigenes and mapped 50,158 (larvae), 48,415 (prepupae) and 46,623 (pupae) of them to the NCBI database. Most differentially expressed genes in the 5th instar larvae/prepupae libraries were relevant to nucleotide synthesis and maintenance of silk gland function. We identified down-regulated transcriptional factors and several genes involved in silk formation in the three libraries and verified the expression pattern of eight genes by qPCR. The developmental- and tissue-specific expression patterns of the fibroin light chain gene showed it was highly expressed during the larval silk-producing stage. We recorded highest expression of this gene in the larval silk gland, compared to other tissues, including midgut, hindgut, epidermis, Malpighian tubes, hemolymph and fat body. These data are a genetic resource to guide selection of key genes that may be targeted for in planta and other gene-silencing technologies for sustainable cotton agriculture. PMID:26352931

Silk Gland Gene Expression during Larval-Pupal Transition in the Cotton Leaf Roller Sylepta derogata (Lepidoptera: Pyralidae).

PubMed

Su, Honghua; Cheng, Yuming; Wang, Zhongyang; Li, Zhong; Stanley, David; Yang, Yizhong

2015-01-01

The cotton leaf roller, Sylepta derogata, is a silk-producing insect pest. While young larvae feed on the underside of leaves, the older ones roll cotton leaves and feed on the leaf edges, which defoliates cotton plants. The larvae produce silk to stabilize the rolled leaf and to balloon from used to new leaves. Despite the significance of silk in the biology of pest insect species, there is virtually no information on the genes involved in their silk production. This is a substantial knowledge gap because some of these genes may be valuable targets for developing molecular pest management technologies. We addressed the gap by posing the hypothesis that silk gland gene expression changes during the transition from larvae to pupae. We tested our hypothesis using RNA-seq to investigate changes in silk gland gene expression at three developmental stages, 5th instar larvae (silk producing; 15,445,926 clean reads), prepupae (reduced silk producing; 13,758,154) and pupae (beyond silk producing; 16,787,792). We recorded 60,298 unigenes and mapped 50,158 (larvae), 48,415 (prepupae) and 46,623 (pupae) of them to the NCBI database. Most differentially expressed genes in the 5th instar larvae/prepupae libraries were relevant to nucleotide synthesis and maintenance of silk gland function. We identified down-regulated transcriptional factors and several genes involved in silk formation in the three libraries and verified the expression pattern of eight genes by qPCR. The developmental- and tissue-specific expression patterns of the fibroin light chain gene showed it was highly expressed during the larval silk-producing stage. We recorded highest expression of this gene in the larval silk gland, compared to other tissues, including midgut, hindgut, epidermis, Malpighian tubes, hemolymph and fat body. These data are a genetic resource to guide selection of key genes that may be targeted for in planta and other gene-silencing technologies for sustainable cotton agriculture.

Genome-Wide Transcriptional Reorganization Associated with Senescence-to-Immortality Switch during Human Hepatocellular Carcinogenesis

PubMed Central

Konu, Ozlen; Yuzugullu, Haluk; Gursoy-Yuzugullu, Ozge; Ozturk, Nuri; Ozen, Cigdem; Ozdag, Hilal; Erdal, Esra; Karademir, Sedat; Sagol, Ozgul; Mizrak, Dilsa; Bozkaya, Hakan; Ilk, Hakki Gokhan; Ilk, Ozlem; Bilen, Biter; Cetin-Atalay, Rengul; Akar, Nejat; Ozturk, Mehmet

2013-01-01

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become “immortal”) by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism. PMID:23691139

[Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

PubMed

Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

2014-09-01

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

Branched-chain amino acids in metabolic signalling and insulin resistance

PubMed Central

Lynch, Christopher J.; Adams, Sean H.

2015-01-01

Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM. PMID:25287287

Effects of a meal rich in medium-chain saturated fat on postprandial lipemia in relatives of type 2 diabetics.

PubMed

Pietraszek, Anna; Hermansen, Kjeld; Pedersen, Steen B; Langdahl, Bente L; Holst, Jens J; Gregersen, Søren

2013-01-01

Patients with type 2 diabetes and their relatives (REL) have increased risk for cardiovascular disease (CVD). Postprandial triglyceridemia (PPL), which is influenced by diet, is an independent risk factor for CVD. Little is known about the effects of medium-chain saturated fatty acids (medium-chain SFA) on PPL and gene expression in REL. The objective of this study was to test the hypothesis that medium-chain SFA cause larger PPL response in REL compared with controls (CON) and have a differential effect on circulating incretins and ghrelin and gene expression in muscle and adipose tissue in REL and CON. Seventeen REL and 17 CON received a fat-rich meal (79 energy percent from fat) based on medium-chain SFA (coconut oil). Plasma concentrations of triglycerides (TG), free-fatty acids, insulin, glucose, glucagon-like peptide-1, glucose-dependent insulintropic peptide, and ghrelin were measured before and during 240 min postprandially. Muscle and adipose tissue biopsies were taken at baseline and after the test meal. After the test meal, REL had a higher plasma TG response (P = 0.002) and a tendency toward higher insulin response (P = 0.100). A number of genes were upregulated in response to the meal rich in medium-chain SFA in CON, but not in REL. A meal high in medium-chain SFA resulted in larger PPL response in REL than in CON. It remains to be clarified whether this can be reproduced by a pure medium-chain fat (MCT) load. The meal exerted a differential effect on gene expression in muscle, but not adipose tissue, of REL compared with CON. Copyright © 2013 Elsevier Inc. All rights reserved.

Microarray analysis of gene expression after electrical stimulation of the dura mater surrounding the superior sagittal sinus in conscious adult rats.

PubMed

Jiang, Lei; Dong, Zhao; Li, Fengpeng; Liu, Ruozhuo; Qiu, Enchao; Wang, Xiaolin; Yu, Shengyuan

2014-01-01

The molecular and cellular origins of migraine headache are among the most complex problems in contemporary neurology. Up to now the pathogenesis of migraine still remains unclearly defined. The objective of this study was to explore new factors that may be related to the mechanism of migraine. The present study performed a comprehensive analysis of gene expression in the trigeminal nucleus caudalis induced by electrical stimulation of dura mater surrounding the superior sagittal sinus in conscious rats using microarray analysis followed by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR) verification. Student's two sample t-test was employed when two groups were compared. A P value <0.05 was considered to be statistically significant. Comparing the placebo and the electrical stimulation groups, 40 genes were determined to be significantly differentially expressed. These significantly differentially expressed genes were involved in many pathways, including transporter activity, tryptophan metabolism, G protein signaling, kinase activity, actin binding, signal transducer activity, anion transport, protein folding, enzyme inhibitor activity, coenzyme metabolism, binding, ion transport, cell adhesion, metal ion transport, oxidoreductase activity, mitochondrion function, and others. Most of the genes were involved in more than 2 pathways. Of particular interest is the up-regulation of Phactr3 and Akap5 and the down-regulation of Kdr. These findings may provide important clues for a better understanding of the molecular mechanism of migraine.

Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

PubMed

Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

2014-06-01

Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

PubMed

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

PubMed Central

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

Major COL4A5 gene rearrangements in patients with juvenile type Alport syndrome

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

Renieri, A.; Galli, L.; Bruttini, M.

1995-11-20

Mutations in the COL4A5 gene, which encodes the {alpha}5 chain of type IV collagen, are found in a large fraction of patients with X-linked Alport syndrome. The recently discovered COL4A6, tightly linked and highly homologous to COL4A5, represents a second candidate gene for Alport syndrome. We analyzed 177 Italian Alport syndrome families by Southern blotting using cDNA probes from both COL4A5 and COL4A6. Nine unrelated families, accounting for 5% of the cases, were found to have a rearrangement in COL4A5. No rearrangements were found in COL4A6, with the exception of a deletion encompassing the 5{prime} ends of both COL4A5 andmore » COL4A6 genes in a patient with Alport syndrome and leiomyomatosis. COL4A5 rearrangements were all intragenic and included 1 duplication and 7 deletions. Polymerase chain reaction (PCR) analysis was carried out to characterize deletion and duplication boundaries and to predict the resulting protein abnormality. The two smallest deletions involved a single exon (exons 17 and 40, respectively), while the largest ones spanned exons 1 to 36. The clinical phenotype of patients in whom a rearrangement in COL4A5 was detected was severe, with progression to end-stage renal failure in juvenile age and hypoacusis occurring in most cases. These data have some important implications in the diagnosis of patients with Alport syndrome. 34 refs., 3 figs., 1 tab.« less

Mutations in TET2 and DNMT3A genes are associated with changes in global and gene-specific methylation in acute myeloid leukemia.

PubMed

Ponciano-Gómez, Alberto; Martínez-Tovar, Adolfo; Vela-Ojeda, Jorge; Olarte-Carrillo, Irma; Centeno-Cruz, Federico; Garrido, Efraín

2017-10-01

Acute myeloid leukemia is characterized by its high biological and clinical heterogeneity, which represents an important barrier for a precise disease classification and accurate therapy. While epigenetic aberrations play a pivotal role in acute myeloid leukemia pathophysiology, molecular signatures such as change in the DNA methylation patterns and genetic mutations in enzymes needed to the methylation process can also be helpful for classifying acute myeloid leukemia. Our study aims to unveil the relevance of DNMT3A and TET2 genes in global and specific methylation patterns in acute myeloid leukemia. Peripheral blood samples from 110 untreated patients with acute myeloid leukemia and 15 healthy control individuals were collected. Global 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from peripheral blood leukocytes were measured by using the MethylFlashTM Quantification kits. DNMT3A and TET2 expression levels were evaluated by real-time quantitative polymerase chain reaction. The R882A hotspot of DNMT3A and exons 6-10 of TET2 were amplified by polymerase chain reaction and sequenced using the Sanger method. Methylation patterns of 16 gene promoters were evaluated by pyrosequencing after treating DNA with sodium bisulfite, and their transcriptional products were measured by real-time quantitative polymerase chain reaction.Here, we demonstrate altered levels of 5-methylcytosine and 5-hydroxymethylcytosine and highly variable transcript levels of DNMT3A and TET2 in peripheral blood leukocytes from acute myeloid leukemia patients. We found a mutation prevalence of 2.7% for DNMT3A and 11.8% for TET2 in the Mexican population with this disease. The average overall survival of acute myeloid leukemia patients with DNMT3A mutations was only 4 months. In addition, we showed that mutations in DNMT3A and TET2 may cause irregular DNA methylation patterns and transcriptional expression levels in 16 genes known to be involved in acute myeloid leukemia pathogenesis. Our findings suggest that alterations in DNMT3A and TET2 may be associated with acute myeloid leukemia prognosis. Furthermore, alterations in these enzymes affect normal methylation patterns in acute myeloid leukemia- specific genes, which in turn, may influence patient survival.

Identification of floral genes for sex determination in Calamus palustris Griff. by using suppression subtractive hybridization.

PubMed

Ng, C Y; Wickneswari, R; Choong, C Y

2014-08-07

Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.

A Recessive Contiguous Gene Deletion of Chromosome 2p16 Associated with Cystinuria and a Mitochondrial Disease

PubMed Central

Parvari, Ruti; Brodyansky, Irena; Elpeleg, Orly; Moses, Shimon; Landau, Daniel; Hershkovitz, Eli

2001-01-01

Deletions ranging from 100 Kb to 1 Mb—too small to be detected under the microscope—may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cβ gene (PP2Cβ), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes’ products, one of which may be essential for the synthesis of mitochondrial encoded proteins. PMID:11524703

Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor.

PubMed

Kajita, Shinya; Sugawara, Shinsuke; Miyazaki, Yasumasa; Nakamura, Masaya; Katayama, Yoshihiro; Shishido, Kazuo; Iimura, Yosuke

2004-12-01

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.

Growth arrest specific gene 2 in tilapia (Oreochromis niloticus): molecular characterization and functional analysis under low-temperature stress.

PubMed

Yang, ChangGeng; Wu, Fan; Lu, Xing; Jiang, Ming; Liu, Wei; Yu, Lijuan; Tian, Juan; Wen, Hua

2017-07-17

Growth arrest specific 2 (gas2) gene is a component of the microfilament system that plays a major role in the cell cycle, regulation of microfilaments, and cell morphology during apoptotic processes. However, little information is available on fish gas2. In this study, the tilapia (Oreochromis niloticus) gas2 gene was cloned and characterized for the first time. The open reading frame was 1020 bp, encoding 340 amino acids; the 5'-untranslated region (UTR) was 140 bp and the 3'-UTR was 70 bp, with a poly (A) tail. The highest promoter activity occurred in the regulatory region (-3000 to -2400 bp). The Gas2-GFP fusion protein was distributed within the cytoplasm. Quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed that gas2 gene expression levels in the liver, muscle, and brain were clearly affected by low temperature stress. The results of gas2 RNAi showed decreased expression of the gas2 and P53 genes. These results suggest that the tilapia gas2 gene may be involved in low temperature stress-induced apoptosis.

Complex inheritance in Pulmonary Arterial Hypertension patients with several mutations

PubMed Central

Pousada, Guillermo; Baloira, Adolfo; Valverde, Diana

2016-01-01

Pulmonary Arterial Hypertension (PAH) is a rare and progressive disease with low incidence and prevalence, and elevated mortality. PAH is characterized by increased mean pulmonary artery pressure. The aim of this study was to analyse patients with combined mutations in BMPR2, ACVRL1, ENG and KCNA5 genes and to establish a genotype-phenotype correlation. Major genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Genotype-phenotype correlation was performed. Fifty-seven (28 idiopathic PAH, 29 associated PAH group I) were included. Several mutations in different genes, classified as pathogenic by in silico analysis, were present in 26% of PAH patients. The most commonly involved gene was BMPR2 (12 patients) followed by ENG gene (9 patients). ACVRL1 and KCNA5 genes showed very low incidence of mutations (5 and 1 patients, respectively). Genotype-phenotype correlation showed statistically significant differences for gender (p = 0.045), age at diagnosis (p = 0.035), pulmonary vascular resistance (p = 0.030), cardiac index (p = 0.035) and absence of response to treatment (p = 0.011). PAH is consequence of a heterogeneous constellation of genetic arrangements. Patients with several pathogenic mutations seem to display a more severe phenotype. PMID:27630060

MSX1 gene variant - its presence in tooth absence - a case control genetic study.

PubMed

Reddy, Naveen Admala; Adusumilli, Gopinath; Devanna, Raghu; Pichai, Saravanan; Rohra, Mayur Gobindram; Arjunan, Sharmila

2013-10-01

Non Syndromic tooth agenesis is a congenital anomaly with significant medical, psychological and social ramifications. There is sufficient evidence to hypothesize that locus for this condition can be identified by candidate genes. The aim of this study was to test whether MSX1 671 T>C gene variant was involved in etiology of Non Syndromic tooth agenesis in Raichur Patients. Blood samples were collected with informed consent from 50 subjects having Non Syndromic tooth agenesis and 50 controls. Genomic DNA was extracted from the blood samples, Polymerase Chain Reaction was performed (PCR) and Restriction Fragment Length Polymorphism (RFLP) was performed for digestion products that were evaluated. The RESULTS showed positive correlation between MSX1671 T>C gene variant and Non Syndromic tooth agenesis in Raichur Patients. MSX1 671 T>C gene variant may be a good screening marker for Non Syndromic tooth agenesis in Raichur Patients . How to cite this article:Reddy NA, Adusumilli G, Devanna R, Pichai S, Rohra MG, Arjunan S. Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study. J Int Oral Health 2013; 5(5):20-6.

Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study

PubMed Central

Reddy, Naveen Admala; Adusumilli, Gopinath; Devanna, Raghu; Pichai, Saravanan; Rohra, Mayur Gobindram; Arjunan, Sharmila

2013-01-01

Background: Non Syndromic tooth agenesis is a congenital anomaly with significant medical, psychological and social ramifications. There is sufficient evidence to hypothesize that locus for this condition can be identified by candidate genes. The aim of this study was to test whether MSX1 671 T>C gene variant was involved in etiology of Non Syndromic tooth agenesis in Raichur Patients. Materials & Methods: Blood samples were collected with informed consent from 50 subjects having Non Syndromic tooth agenesis and 50 controls. Genomic DNA was extracted from the blood samples, Polymerase Chain Reaction was performed (PCR) and Restriction Fragment Length Polymorphism (RFLP) was performed for digestion products that were evaluated. Results: The Results showed positive correlation between MSX1671 T>C gene variant and Non Syndromic tooth agenesis in Raichur Patients. Conclusion: MSX1 671 T>C gene variant may be a good screening marker for Non Syndromic tooth agenesis in Raichur Patients . How to cite this article:Reddy NA, Adusumilli G, Devanna R, Pichai S, Rohra MG, Arjunan S. Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study. J Int Oral Health 2013; 5(5):20-6. PMID:24324300

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

PubMed Central

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

PubMed

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Antibody Light-Chain-Restricted Recognition of the Site of Immune Pressure in the RV144 HIV-1 Vaccine Trial Is Phylogenetically Conserved

DOE PAGES

Wiehe, Kevin; Easterhoff, David; Luo, Kan; ...

2014-11-29

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognitionmore » prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. In conclusion, these data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.« less

Antibody Light-Chain-Restricted Recognition of the Site of Immune Pressure in the RV144 HIV-1 Vaccine Trial Is Phylogenetically Conserved

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

Wiehe, Kevin; Easterhoff, David; Luo, Kan

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognitionmore » prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. In conclusion, these data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.« less

Food handlers as a link in the chain of transmission of Giardia duodenalis and other protozoa in public schools in southern Brazil.

PubMed

Colli, Cristiane Maria; Bezagio, Renata Coltro; Nishi, Letícia; Ferreira, Érika Cristina; Falavigna-Guilherme, Ana Lúcia; Gomes, Mônica Lúcia

2015-09-01

Food handlers (FHs) may facilitate transmission and dissemination of pathogens. The importance of FHs as a link in the epidemiological chain of transmission of Giardia duodenalis and other intestinal protozoa was assessed. Fecal and subungual material from 27 FHs were analyzed using parasitological methods. G. duodenalis was identified by direct immunofluorescence and genotyped by PCR-RFLP for the bg and gdh genes, and gdh was sequenced. At least one protozoan was detected in 30% (8/27) of the FHs and G. duodenalis (19%; 5/27) was the most common species. The AII and BIV genotypes were found in 20% (1/5) and 60% (3/5) of FHs infected with G. duodenalis, respectively. FHs can be involved in the chain of transmission of G. duodenalis and other protozoa. KJ741310 - KJ741313. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes.

PubMed

Makeyev, Eugene V; Bamford, Dennis H

2002-12-01

Recent genetic data suggest that proteins homologous to a plant RNA-dependent RNA polymerase (RdRP) play a central role in posttranscriptional gene silencing (PTGS) in many organisms. We show here that purified recombinant protein QDE-1, a genetic component of PTGS ("quelling") in the fungus Neurospora crassa, possesses RNA polymerase activity in vitro. The full-length enzyme and its enzymatically active C-terminal fragment perform two different reactions on single-stranded RNA templates, synthesizing either extensive RNA chains that form template-length duplexes or approximately 9-21-mer complementary RNA oligonucleotides scattered along the entire template. QDE-1 supports both de novo and primer-dependent initiation mechanisms. These results suggest that several distinct activities of cell-encoded RdRPs can be employed for efficient PTGS in vivo.

Molecular cloning and characterization of adult Sparus aurata hemoglobin genes.

PubMed

Campo, Salvatore; Nastasi, Giancarlo; Fedeli, Donatella; D'Ascola, Angela; Campo, Giuseppe M; Avenoso, Angela; Ferlazzo, Alida; Calatroni, Alberto; Falcioni, Giancarlo

2010-04-01

Among Teleosts, Sparus aurata occupies a prominent place in the gastronomic and economic fields of the Mediterranean basin and other geographic districts. The knowledge of its molecular structures and functional features, such as hemoglobin, may be helpful to understand the adaptive biochemical mechanisms that allow this fish to live under extreme conditions, including fish farming. In Sparus aurata red blood cells two different alpha and one beta hemoglobin genes have been identified. The alpha1 gene codifies a putative protein of 144 amino acids, the alpha2 gene produces a protein of 143 amino acids, and the beta gene encodes a chain of 148 amino acids. Comparative analysis of various hemoglobins indicates that allosteric regulation can be modified by the substitution of one or a few key residues. The comparison of the percentage sequence differences for alpha and beta chains in fishes indicates that evolutionary relationships between different species may be helpful to understand the mechanisms of their differentiation from other vertebrates. Hemoglobin alpha and beta chains of about 50 teleostean temperate and Antarctic fishes were analyzed to build phylogenetic trees using different algorithms: the neighbor-joining method, the maximum likelihood approach, and the Bayesian inference computation. Sparus aurata alpha chains are positioned in a paraphyletic cluster, which includes the same subunit of Chrysophrys auratus and Seriola quinqueradiata, whereas the beta chain is on an homophyletic branch with that of Chrysophrys auratus. Therefore, the phylogenetic approach suggests that both Sparus aurata hemoglobin alpha genes are paralogues and may have derived from a duplication event.

No Association Between Variant N-acetyltransferase Genes, Cigarette Smoking and Prostate Cancer Susceptibility Among Men of African Descent

PubMed Central

Kidd, La Creis Renee; VanCleave, Tiva T.; Doll, Mark A.; Srivastava, Daya S.; Thacker, Brandon; Komolafe, Oyeyemi; Pihur, Vasyl; Brock, Guy N.; Hein, David W.

2011-01-01

Objective We evaluated the individual and combination effects of NAT1, NAT2 and tobacco smoking in a case-control study of 219 incident prostate cancer (PCa) cases and 555 disease-free men. Methods Allelic discriminations for 15 NAT1 and NAT2 loci were detected in germ-line DNA samples using Taqman polymerase chain reaction (PCR) assays. Single gene, gene-gene and gene-smoking interactions were analyzed using logistic regression models and multi-factor dimensionality reduction (MDR) adjusted for age and subpopulation stratification. MDR involves a rigorous algorithm that has ample statistical power to assess and visualize gene-gene and gene-environment interactions using relatively small samples sizes (i.e., 200 cases and 200 controls). Results Despite the relatively high prevalence of NAT1*10/*10 (40.1%), NAT2 slow (30.6%), and NAT2 very slow acetylator genotypes (10.1%) among our study participants, these putative risk factors did not individually or jointly increase PCa risk among all subjects or a subset analysis restricted to tobacco smokers. Conclusion Our data do not support the use of N-acetyltransferase genetic susceptibilities as PCa risk factors among men of African descent; however, subsequent studies in larger sample populations are needed to confirm this finding. PMID:21709725

Altered Expression of Diabetes-Related Genes in Alzheimer's Disease Brains: The Hisayama Study

PubMed Central

Hokama, Masaaki; Oka, Sugako; Leon, Julio; Ninomiya, Toshiharu; Honda, Hiroyuki; Sasaki, Kensuke; Iwaki, Toru; Ohara, Tomoyuki; Sasaki, Tomio; LaFerla, Frank M.; Kiyohara, Yutaka; Nakabeppu, Yusaku

2014-01-01

Diabetes mellitus (DM) is considered to be a risk factor for dementia including Alzheimer's disease (AD). However, the molecular mechanism underlying this risk is not well understood. We examined gene expression profiles in postmortem human brains donated for the Hisayama study. Three-way analysis of variance of microarray data from frontal cortex, temporal cortex, and hippocampus was performed with the presence/absence of AD and vascular dementia, and sex, as factors. Comparative analyses of expression changes in the brains of AD patients and a mouse model of AD were also performed. Relevant changes in gene expression identified by microarray analysis were validated by quantitative real-time reverse-transcription polymerase chain reaction and western blotting. The hippocampi of AD brains showed the most significant alteration in gene expression profile. Genes involved in noninsulin-dependent DM and obesity were significantly altered in both AD brains and the AD mouse model, as were genes related to psychiatric disorders and AD. The alterations in the expression profiles of DM-related genes in AD brains were independent of peripheral DM-related abnormalities. These results indicate that altered expression of genes related to DM in AD brains is a result of AD pathology, which may thereby be exacerbated by peripheral insulin resistance or DM. PMID:23595620

[Up regulation of phenylacetate to glioma homeobox gene expression].

PubMed

Tian, Yu; Yang, Chaohua; Zhao, Conghai

2002-03-01

Even though phenylacetate (PA) bas been shown to inhibit the growth and induce differentiation in rat C6 glioma cell line, its mechanisms are still poorly understood. This study is aimed to identify which Hox gene is related to glioma and to observe the change in expression on mRNA level as treated by phenylasetate. Twenty-two kinds of Hox gene were divided into 3 groups according to their primer sequence. Semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) was used to investigate the mRNA expression of Hox gene groups and some Hox gene in rat C6 glioma cell line following differentiation induced by PA. The level of Hox gene expression was expressed as ratio expression rate (RER) of Hox gene/beta-actin according to computer image analysis and the difference between C6 cells and PA treated C6 cells was analyzed by student t-test. It was found that Hox genes matching to primers P2 were mildly expressed in C6 cells and the expression of HoxB2 mRNA was significantly up-regulated in PA treated C6 cells (P < 0.001). The weak expression of HoxB2 may be involved in glioma origin and the mechanisms of PA action are correlated with transcription process in the glioma cells.

CpG site hypermethylation of E-cadherin and Connexin26 genes in hepatocellular carcinomas induced by a choline-deficient L-Amino Acid-defined diet in rats.

PubMed

Tsujiuchi, Toshifumi; Shimizu, Kyoko; Itsuzaki, Yumi; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Honoki, Kanya

2007-04-01

We investigated DNA methylation patterns of E-cadherin and Connexin26 (Cx26) genes in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-Amino Acid-defined (CDAA) diet. Six-wks-old F344 male rats were continuously fed with a CDAA diet for 75 wks, and were then killed. A total of five HCCs were obtained, and genomic DNA was extracted from each HCC for assessment of methylation status in the 5' upstream regions of E-cadherin and Cx26 genes by bisulfite sequencing, comparing to two normal liver tissues. The five HCCs showed highly methylated E-cadherin and Cx26 genes, while these genes in two normal liver tissues were all unmethylated. For analysis of gene expression, real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR) was performed. Expressions of E-cadherin and Cx26 genes were significantly reduced in the five HCCs (P < 0.0001 and P < 0.001, respectively) compared to normal liver tissues, correlating with their methylation statuses. These results suggested that hypermethylation of E-cadherin and Cx26 genes may be involved in the development of HCCs induced by a CDAA diet in rats.

A large homozygous deletion in the SAMHD1 gene causes atypical Aicardi–Goutiéres syndrome associated with mtDNA deletions

PubMed Central

Leshinsky-Silver, Esther; Malinger, Gustavo; Ben-Sira, Liat; Kidron, Dvora; Cohen, Sarit; Inbar, Shani; Bezaleli, Tali; Levine, Arie; Vinkler, Chana; Lev, Dorit; Lerman-Sagie, Tally

2011-01-01

Aicardi–Goutiéres syndrome (AGS) is a genetic neurodegenerative disorder with clinical symptoms mimicking a congenital viral infection. Five causative genes have been described: three prime repair exonuclease1 (TREX1), ribonucleases H2A, B and C, and most recently SAM domain and HD domain 1 (SAMHD1). We performed a detailed clinical and molecular characterization of a family with autosomal recessive neurodegenerative disorder showing white matter destruction and calcifications, presenting in utero and associated with multiple mtDNA deletions. A muscle biopsy was normal and did not show any evidence of respiratory chain dysfunction. Southern blot analysis of tissue from a living child and affected fetuses demonstrated multiple mtDNA deletions. Molecular analysis of genes involved in mtDNA synthesis and maintenance (POLGα, POLGβ, Twinkle, ANT1, TK2, SUCLA1 and DGOUK) revealed normal sequences. Sequencing of TREX1 and ribonucleases H2A, B and C failed to reveal any mutations. Whole-genome homozygosity mapping revealed a candidate region containing the SAMHD1 gene. Sequencing of the gene in the affected child and two affected fetuses revealed a large deletion (9 kb), spanning the promoter, exon1 and intron 1. The parents were found to be heterozygous for this deletion. The identification of a homozygous large deletion in the SAMHD1 gene causing atypical AGS with multiple mtDNA deletions may add information regarding the involvement of mitochondria in self-activation of innate immunity by cell intrinsic components. PMID:21102625

Involvement of the mitfa gene in the development of pigment cell in Japanese ornamental (Koi) carp (Cyprinus carpio L.).

PubMed

Liu, J H; Wen, S; Luo, C; Zhang, Y Q; Tao, M; Wang, D W; Deng, S M; Xiao, Y M

2015-03-31

A colored phenotype is an important feature of ornamental fish. In mammals, microphthalmia-associated transcription factor (MITF) was found to regulate the development of melanocytes. In this study, the mitfa cDNA was first cloned from the Japanese ornamental (Koi) carp (Cyprinus carpio L.), an important ornamental freshwater fish. The full-length cDNA of the mitfa gene contains 1634 bp, coding for 412 amino acids in Koi. The identity degree of mitfa amino acid sequences between the Koi carp and zebrafish is 92.9%. We tested the expression of the mitfa gene in several varieties of Koi using reverse transcription-polymerase chain reaction and found that the mitfa gene is highly expressed in the skin tissues of the Taisho sanke and the Procypris merus. Interestingly, the mitfa gene was also expressed in the Kohaku and Yamabaki ogon, although melanocytes were not observed in the skin. Koi carp embryos were transparent and colorless, while after hatching, different types of pigment cells successively emerged in a fixed order. In Taisho sanke, melanocytes first appeared in the trunk at approximately 12 days of age. Subsequently, there was a large area of melanocytes by 30 days of age. The expression level of the mitfa mRNA was low in early embryos and newly hatched larvae, and increased to high levels in 30-day-old fry. The results show that the mitfa gene is involved in regulating fish body color in the development of both melanocytes and pigment cells.

Dynamic gene expression response to altered gravity in human T cells.

PubMed

Thiel, Cora S; Hauschild, Swantje; Huge, Andreas; Tauber, Svantje; Lauber, Beatrice A; Polzer, Jennifer; Paulsen, Katrin; Lier, Hartwin; Engelmann, Frank; Schmitz, Burkhard; Schütte, Andreas; Layer, Liliana E; Ullrich, Oliver

2017-07-12

We investigated the dynamics of immediate and initial gene expression response to different gravitational environments in human Jurkat T lymphocytic cells and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. We used the Affymetrix GeneChip® Human Transcriptome Array 2.0 containing 44,699 protein coding genes and 22,829 non-protein coding genes and performed the experiments during a parabolic flight and a suborbital ballistic rocket mission to cross-validate gravity-regulated gene expression through independent research platforms and different sets of control experiments to exclude other factors than alteration of gravity. We found that gene expression in human T cells rapidly responded to altered gravity in the time frame of 20 s and 5 min. The initial response to microgravity involved mostly regulatory RNAs. We identified three gravity-regulated genes which could be cross-validated in both completely independent experiment missions: ATP6V1A/D, a vacuolar H + -ATPase (V-ATPase) responsible for acidification during bone resorption, IGHD3-3/IGHD3-10, diversity genes of the immunoglobulin heavy-chain locus participating in V(D)J recombination, and LINC00837, a long intergenic non-protein coding RNA. Due to the extensive and rapid alteration of gene expression associated with regulatory RNAs, we conclude that human cells are equipped with a robust and efficient adaptation potential when challenged with altered gravitational environments.

Effects of the Transforming Growth Factor Beta Signaling Pathway on the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells

PubMed Central

Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Li, Dong; Zhang, Wenhui; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Wang, Man; Wang, Kehua

2016-01-01

Abstract The objectives of the present study were to screen for key gene and signaling pathways involved in the production of male germ cells in poultry and to investigate the effects of the transforming growth factor beta (TGF-β) signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into male germ cells. The ESCs, primordial germ cells, and spermatogonial stem cells (SSCs) were sorted using flow cytometry for RNA sequencing (RNA-seq) technology. Male chicken ESCs were induced using 40 ng/mL of bone morphogenetic protein 4 (BMP4). The effects of the TGF-β signaling pathway on the production of chicken SSCs were confirmed by morphology, quantitative real-time polymerase chain reaction, and immunocytochemistry. One hundred seventy-three key genes relevant to development, differentiation, and metabolism and 20 signaling pathways involved in cell reproduction, differentiation, and signal transduction were identified by RNA-seq. The germ cells formed agglomerates and increased in number 14 days after induction by BMP4. During the induction process, the ESCs, Nanog, and Sox2 marker gene expression levels decreased, whereas expression of the germ cell-specific genes Stra8, Dazl, integrin-α6, and c-kit increased. The results indicated that the TGF-β signaling pathway participated in the differentiation of chicken ESCs into male germ cells. PMID:27906584

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi

PubMed Central

Johnson, Aaron N.; Mokalled, Mayssa H.; Valera, Juliana M.; Poss, Kenneth D.; Olson, Eric N.

2013-01-01

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression. PMID:23942517

Life-span extension by dietary restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C. elegans.

PubMed

Park, Sang-Kyu; Link, Christopher D; Johnson, Thomas E

2010-02-01

Recent studies have shown that the rate of aging can be modulated by diverse interventions. Dietary restriction is the most widely used intervention to promote longevity; however, the mechanisms underlying the effect of dietary restriction remain elusive. In a previous study, we identified two novel genes, nlp-7 and cup-4, required for normal longevity in Caenorhabditis elegans. nlp-7 is one of a set of neuropeptide-like protein genes; cup-4 encodes an ion-channel involved in endocytosis by coelomocytes. Here, we assess whether nlp-7 and cup-4 mediate longevity increases by dietary restriction. RNAi of nlp-7 or cup-4 significantly reduces the life span of the eat-2 mutant, a genetic model of dietary restriction, but has no effect on the life span of long-lived mutants resulting from reduced insulin/IGF-1 signaling or dysfunction of the mitochondrial electron transport chain. The life-span extension observed in wild-type N2 worms by dietary restriction using bacterial dilution is prevented significantly in nlp-7 and cup-4 mutants. RNAi knockdown of genes encoding candidate receptors of NLP-7 and genes involved in endocytosis by coelomocytes also specifically shorten the life span of the eat-2 mutant. We conclude that two novel pathways, NLP-7 signaling and endocytosis by coelomocytes, are required for life extension under dietary restriction in C. elegans.

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

NASA Technical Reports Server (NTRS)

Criswell, D. S.; Carson, J. A.; Booth, F. W.

1996-01-01

Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.

Orthogonal Polynomials Associated with Complementary Chain Sequences

NASA Astrophysics Data System (ADS)

Behera, Kiran Kumar; Sri Ranga, A.; Swaminathan, A.

2016-07-01

Using the minimal parameter sequence of a given chain sequence, we introduce the concept of complementary chain sequences, which we view as perturbations of chain sequences. Using the relation between these complementary chain sequences and the corresponding Verblunsky coefficients, the para-orthogonal polynomials and the associated Szegő polynomials are analyzed. Two illustrations, one involving Gaussian hypergeometric functions and the other involving Carathéodory functions are also provided. A connection between these two illustrations by means of complementary chain sequences is also observed.

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

Martinell, J.; Whitney, J.B.; Popp, R.A.

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hba/sup th-J/, appear to be true animal models of human thalassemia. Expression of the ..cap alpha..-globin genes in three stocks of mice, each one heterozygous for one of the ..cap alpha..-globin mutations, was examined at the polypeptide, RNA, and DNA levels. ..cap alpha..-globin polypeptide chains, relative to ..gamma..-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of ..cap alpha..-globin to ..gamma..-globin RNA sequences are also 75 to 80% normal, exactly reflecting the ..cap alpha..-globin to ..gamma..-globin chain ratios. In the case ofmore » mutant 352HB, at least one ..cap alpha..-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their ..cap alpha..-globin genes.« less

Nested methylation-specific polymerase chain reaction cancer detection method

DOEpatents

Belinsky, Steven A [Albuquerque, NM; Palmisano, William A [Edgewood, NM

2007-05-08

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases

PubMed Central

Martín, Juan F.; Liras, Paloma

2017-01-01

The clavine alkaloids produced by the fungi of the Aspergillaceae and Arthrodermatacea families differ from the ergot alkaloids produced by Claviceps and Neotyphodium. The clavine alkaloids lack the extensive peptide chain modifications that occur in lysergic acid derived ergot alkaloids. Both clavine and ergot alkaloids arise from the condensation of tryptophan and dimethylallylpyrophosphate by the action of the dimethylallyltryptophan synthase. The first five steps of the biosynthetic pathway that convert tryptophan and dimethylallyl-pyrophosphate (DMA-PP) in chanoclavine-1-aldehyde are common to both clavine and ergot alkaloids. The biosynthesis of ergot alkaloids has been extensively studied and is not considered in this article. We focus this review on recent advances in the gene clusters for clavine alkaloids in the species of Penicillium, Aspergillus (Neosartorya), Arthroderma and Trychophyton and the enzymes encoded by them. The final products of the clavine alkaloids pathways derive from the tetracyclic ergoline ring, which is modified by late enzymes, including a reverse type prenyltransferase, P450 monooxygenases and acetyltransferases. In Aspergillus japonicus, a α-ketoglutarate and Fe2+-dependent dioxygenase is involved in the cyclization of a festuclavine-like unknown type intermediate into cycloclavine. Related dioxygenases occur in the biosynthetic gene clusters of ergot alkaloids in Claviceps purpurea and also in the clavine clusters in Penicillium species. The final products of the clavine alkaloid pathway in these fungi differ from each other depending on the late biosynthetic enzymes involved. An important difference between clavine and ergot alkaloid pathways is that clavine producers lack the enzyme CloA, a P450 monooxygenase, involved in one of the steps of the conversion of chanoclavine-1-aldehyde into lysergic acid. Bioinformatic analysis of the sequenced genomes of the Aspergillaceae and Arthrodermataceae fungi showed the presence of clavine gene clusters in Arthroderma species, Penicillium roqueforti, Penicillium commune, Penicillium camemberti, Penicillium expansum, Penicillium steckii and Penicillium griseofulvum. Analysis of the gene clusters in several clavine alkaloid producers indicates that there are gene gains, gene losses and gene rearrangements. These findings may be explained by a divergent evolution of the gene clusters of ergot and clavine alkaloids from a common ancestral progenitor six genes cluster although horizontal gene transfer of some specific genes may have occurred more recently. PMID:29186777

Identification and expression of the WRKY transcription factors of Carica papaya in response to abiotic and biotic stresses.

PubMed

Pan, Lin-Jie; Jiang, Ling

2014-03-01

The WRKY transcription factor (TF) plays a very important role in the response of plants to various abiotic and biotic stresses. A local papaya database was built according to the GenBank expressed sequence tag database using the BioEdit software. Fifty-two coding sequences of Carica papaya WRKY TFs were predicted using the tBLASTn tool. The phylogenetic tree of the WRKY proteins was classified. The expression profiles of 13 selected C. papaya WRKY TF genes under stress induction were constructed by quantitative real-time polymerase chain reaction. The expression levels of these WRKY genes in response to 3 abiotic and 2 biotic stresses were evaluated. TF807.3 and TF72.14 are upregulated by low temperature; TF807.3, TF43.76, TF12.199 and TF12.62 are involved in the response to drought stress; TF9.35, TF18.51, TF72.14 and TF12.199 is involved in response to wound; TF12.199, TF807.3, TF21.156 and TF18.51 was induced by PRSV pathogen; TF72.14 and TF43.76 are upregulated by SA. The regulated expression levels of above eight genes normalized against housekeeping gene actin were significant at probability of 0.01 levels. These WRKY TFs could be related to corresponding stress resistance and selected as the candidate genes, especially, the two genes TF807.3 and TF12.199, which were regulated notably by four stresses respectively. This study may provide useful information and candidate genes for the development of transgenic stress tolerant papaya varieties.

Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism.

PubMed

Qi, Zhigang; Smith, Kristina M; Bredeweg, Erin L; Bosnjak, Natasa; Freitag, Michael; Nargang, Frank E

2017-02-09

In Neurospora crassa , blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa , we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome. Copyright © 2017 Qi et al.

Detecting genome-wide gene transcription profiles associated with high pollution burden in the critically endangered European eel.

PubMed

Pujolar, J M; Milan, M; Marino, I A M; Capoccioni, F; Ciccotti, E; Belpaire, C; Covaci, A; Malarvannan, G; Patarnello, T; Bargelloni, L; Zane, L; Maes, G E

2013-05-15

The European eel illustrates an example of a critically endangered fish species strongly affected by human stressors throughout its life cycle, in which pollution is considered to be one of the factors responsible for the decline of the stock. The objective of our study was to better understand the transcriptional response of European eels chronically exposed to pollutants in their natural environment. A total of 42 pre-migrating (silver) female eels from lowly, highly and extremely polluted environments in Belgium and, for comparative purposes, a lowly polluted habitat in Italy were measured for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Multipollutant level of bioaccumulation was linked to their genome-wide gene transcription using an eel-specific array of 14,913 annotated cDNAs. Shared responses to pollutant exposure were observed when comparing the highly polluted site in Belgium with the relatively clean sites in Belgium and Italy. First, an altered pattern of transcription of genes was associated with detoxification, with a novel European eel CYP3A gene and gluthatione S-transferase transcriptionally up-regulated. Second, an altered pattern of transcription of genes associated with the oxidative phosphorylation pathway, with the following genes involved in the generation of ATP being transcriptionally down-regulated in individuals from the highly polluted site: NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, cytochrome c oxidase and ATP synthase. Although we did not measure metabolism directly, seeing that the transcription level of many genes encoding enzymes involved in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated in the highly polluted site suggests that pollutants may have a significant effect on energy metabolism in these fish. Copyright © 2013 Elsevier B.V. All rights reserved.

Single nucleotide polymorphism of CC chemokine ligand 5 promoter gene in recipients may predict the risk of chronic graft-versus-host disease and its severity after allogeneic transplantation.

PubMed

Kim, Dong Hwan; Jung, Hee Du; Lee, Nan Young; Sohn, Sang Kyun

2007-10-15

Leukocyte trafficking, regulated by chemokine ligands and their receptors, involves in the pathogenesis of graft-versus-host disease (GVHD) including CC ligand 5 (CCL5) or CC receptor 5 (CCR5). The current study analyzed the association of acute or chronic GVHD (cGVHD) with the CCR5/CCL5 gene single nucleotide polymorphisms (SNPs) of recipients and donors. We evaluated the SNPs of CCL5 promoter gene at position -28 (rs1800825)/-403 (rs2107538) and CCR5 gene at 59029 (rs1799987) in 72 recipients and donors using polymerase chain reaction/RFLP (Restriction Fragment Length Polymorphism) methods. With a median follow up of 924 days for survivors (range 48-2,360 days), the CG genotype of CCL5 gene at position -28 in recipients was significantly associated with a higher incidence of cGVHD (P=0.004), extensive cGVHD (P=0.038 by Seattle's criteria), and severe grade of cGVHD at presentation (P=0.017 by prognostic grading by Apkek et al.) compared to CC genotype. In terms of haplotype analysis, the recipients with AG haplotype of CCL5 gene also showed a higher incidence of cGVHD (P=0.003), extensive cGVHD (P=0.023), and more severe grade of cGVHD (P=0.020). However, there was no association of CCL5/CCR5 SNPs with acute GVHD. The donors' genotype of CCL5/CCR5 was not associated with the risk of cGVHD. The CCL5 promoter gene polymorphism of recipients was associated with the risk of cGVHD and its severity. The current study suggested an involvement of CCL5 in leukocyte trafficking for the development of cGVHD.

A Novel Approach to Dissect the Abscission Process in Arabidopsis1[C][W][OA

PubMed Central

González-Carranza, Zinnia Haydee; Shahid, Ahmad Ali; Zhang, Li; Liu, Yang; Ninsuwan, Unchalee; Roberts, Jeremy Alan

2012-01-01

Abscission is the consequence of a specialized layer of cells undergoing a complex series of molecular and biochemical events. Analysis of the specific molecular changes associated with abscission is hampered by contamination from neighboring nonseparating tissues. Moreover, studies of abscission frequently involve the examination of events that take place in isolated segments of tissue exposed to nonphysiological concentrations of ethylene or indole-3-acetic acid for protracted periods (more than 24 h) of time. To resolve these problems, we have adopted the use of a transgenic line of Arabidopsis (Arabidopsis thaliana) where the promoter of an abscission-specific polygalacturonase gene (At2g41850/ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2) has been fused to a green fluorescent protein reporter. RNA was extracted from green fluorescent protein-tagged cells, released from abscising floral organs, and used to generate a complementary DNA library. This library was used to probe a microarray, and a population of abscission-related transcripts was studied in detail. Seven novel abscission-related genes were identified, four of which encode proteins of unknown function. Reverse transcription-polymerase chain reaction analyses and promoter fusions to the β-glucuronidase reporter gene confirmed the expression of these genes in the abscission zone and revealed other places of expression during seedling development. Three of these genes were studied further by crossing reporter lines to the abscission mutants inflorescence deficient in abscission (ida) and blade-on-petiole1 (bop1)/bop2 and an IDA-overexpressing line. Phenotypic analysis of an At3g14380 transfer DNA insertion line indicates that this gene plays a functional role in floral organ shedding. This strategy has enabled us to uncover new genes involved in abscission, and their possible contribution to the process is discussed. PMID:22992509

Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei

PubMed Central

He, Xiaoyuan; Zhao, Mingfeng; Chen, Jinyan; Wu, Rimao; Zhang, Jianlei; Cui, Rui; Jiang, Yanyu; Chen, Jie; Cao, Xiaoli; Xing, Yi; Zhang, Yuchen; Meng, Juanxia; Deng, Qi; Sui, Tao

2015-01-01

Objective To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei. Methods The 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level. Results We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. PMID:26308936

Medium Chain Triglycerides enhances exercise endurance through the increased mitochondrial biogenesis and metabolism

PubMed Central

Han, Yi; Xu, Jiping; Li, Zhaoshen

2018-01-01

Medium Chain Triglycerides (MCT) is a dietary supplement and usually used along with medications for treating food absorption disorders including diarrhea, steatorrhea and liver disease. It has been shown that MCT plays a role in lowering weight, and decreasing metabolic syndrome, abdominal obesity and inflammation. However, it is still unknown whether MCT enhances exercise endurance. Here, we demonstrated that MCT containing diet improves high temperature induced exercise performance impairment. We found that MCT up-regulates the expression and protein levels of genes involved in mitochondrial biogenesis and metabolism. Further investigation demonstrated that the increased mitochondrial biogenesis and metabolism is mediated through the activation of Akt and AMPK signaling pathways and inhibition of TGF-β signaling pathway. Collectively, our findings indicate a beneficial effect of dietary MCT in exercise performance through the increase of mitochondrial biogenesis and metabolism. PMID:29420554

[The detection and sequence analysis of the simian T-lymphotrophic retrovirus (STLV-1 Papio) by using the polymerase chain reaction].

PubMed

D'iachenko, A G; Dzhalagoniia, B E; Kapanadze, B I

1993-01-01

The gene amplification technique was used for detection and sequence analysis of STLV-1 Papio proviral DNA. The polymerase chain reaction was performed with a primer pair at tax region of HTLV-1, 7336-7354, sense strand, and 7516-7494, antisense strand. One microgram of DNAs isolated from LUG-4 cells and autopsies was used in a reaction volume of 50 microliters involving 30 cycles of amplifications. The reaction product was blunt-end cloned into pUC19 cut with Smal. The sequence was done with T7-polymerase using 32P-dATR as a label. Our results indicate that STLV-1 Papio provirus is actually present in the cells of a lymphoid cell line and tumor cells of lymphomatous monkeys. There are some differences between STLV-1 Papio and reported sequences of HTLV-1 and STLV-1.

[Pea (Pisum sativum) genes, participating in symbiosis with nitrogen-fixing bacteria. III. Study of the structure of the ENOD12 early nodulin gene for various types of peas using the polymerase chain reaction (PCR)].

PubMed

Kozik, A V; Matvienko, M A; Men', A E; Zalenskiĭ, A O; Tikhonovich, I A

1992-01-01

We have determined the length of early noduline gene ENOD12 in various varieties and lines of pea (Pisum sativum) using the polymerase chain reaction (PCR). It was demonstrated that promoter regions of ENOD12A and ENOD12B genes in line 2150 (Afghanistan) are longer than these in variety "Feltham first". The disparity is 14 bp. When studying these genes in 7 different lines and varieties of pea it was found that ENOD12A gene is more variable in size than the ENOD12B gene. We showed the possibility to analyze the heritage of ENOD12 gene's alleles by using the PCR method.

Artemisinin resistance in rodent malaria - mutation in the AP2 adaptor μ-chain suggests involvement of endocytosis and membrane protein trafficking

PubMed Central

2013-01-01

Background The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. Methods Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared. Results An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the μ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene. Conclusion An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum. PMID:23561245

Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal.

PubMed

Bluhm, B H; Flaherty, J E; Cousin, M A; Woloshuk, C P

2002-12-01

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.

Characterization of circulating microRNA expression in patients with a ventricular septal defect.

PubMed

Li, Dong; Ji, Long; Liu, Lianbo; Liu, Yizhi; Hou, Haifeng; Yu, Kunkun; Sun, Qiang; Zhao, Zhongtang

2014-01-01

Ventricular septal defect (VSD), one of the most common types of congenital heart disease (CHD), results from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in development of CHD. This study was to characterize the expression of miRNAs that might be involved in the development or reflect the consequences of VSD. MiRNA microarray analysis and reverse transcription-polymerase chain reaction (RT-PCR) were employed to determine the miRNA expression profile from 3 patients with VSD and 3 VSD-free controls. 3 target gene databases were employed to predict the target genes of differentially expressed miRNAs. miRNAs that were generally consensus across the three databases were selected and then independently validated using real time PCR in plasma samples from 20 VSD patients and 15 VSD-free controls. Target genes of validated 8 miRNAs were predicted using bioinformatic methods. 36 differentially expressed miRNAs were found in the patients with VSD and the VSD-free controls. Compared with VSD-free controls, expression of 15 miRNAs were up-regulated and 21 miRNAs were downregulated in the VSD group. 15 miRNAs were selected based on database analysis results and expression levels of 8 miRNAs were validated. The results of the real time PCR were consistent with those of the microarray analysis. Gene ontology analysis indicated that the top target genes were mainly related to cardiac right ventricle morphogenesis. NOTCH1, HAND1, ZFPM2, and GATA3 were predicted as targets of hsa-let-7e-5p, hsa-miR-222-3p and hsa-miR-433. We report for the first time the circulating miRNA profile for patients with VSD and showed that 7 miRNAs were downregulated and 1 upregulated when matched to VSD-free controls. Analysis revealed target genes involved in cardiac development were probably regulated by these miRNAs.

Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors.

PubMed

Schildberg, Theresa; Rauh, Juliane; Bretschneider, Henriette; Stiehler, Maik

2013-11-01

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended. © 2013.

Recent developments in altering the fatty acid composition of ruminant-derived foods.

PubMed

Shingfield, K J; Bonnet, M; Scollan, N D

2013-03-01

There is increasing evidence to indicate that nutrition is an important factor involved in the onset and development of several chronic human diseases including cancer, cardiovascular disease (CVD), type II diabetes and obesity. Clinical studies implicate excessive consumption of medium-chain saturated fatty acids (SFA) and trans-fatty acids (TFA) as risk factors for CVD, and in the aetiology of other chronic conditions. Ruminant-derived foods are significant sources of medium-chain SFA and TFA in the human diet, but also provide high-quality protein, essential micronutrients and several bioactive lipids. Altering the fatty acid composition of ruminant-derived foods offers the opportunity to align the consumption of fatty acids in human populations with public health policies without the need for substantial changes in eating habits. Replacing conserved forages with fresh grass or dietary plant oil and oilseed supplements can be used to lower medium-chain and total SFA content and increase cis-9 18:1, total conjugated linoleic acid (CLA), n-3 and n-6 polyunsaturated fatty acids (PUFA) to a variable extent in ruminant milk. However, inclusion of fish oil or marine algae in the ruminant diet results in marginal enrichment of 20- or 22-carbon PUFA in milk. Studies in growing ruminants have confirmed that the same nutritional strategies improve the balance of n-6/n-3 PUFA, and increase CLA and long-chain n-3 PUFA in ruminant meat, but the potential to lower medium-chain and total SFA is limited. Attempts to alter meat and milk fatty acid composition through changes in the diet fed to ruminants are often accompanied by several-fold increases in TFA concentrations. In extreme cases, the distribution of trans 18:1 and 18:2 isomers in ruminant foods may resemble that of partially hydrogenated plant oils. Changes in milk fat or muscle lipid composition in response to diet are now known to be accompanied by tissue-specific alterations in the expression of one or more lipogenic genes. Breed influences both milk and muscle fat content, although recent studies have confirmed the occurrence of genetic variability in transcript abundance and activity of enzymes involved in lipid synthesis and identified polymorphisms for several key lipogenic genes in lactating and growing cattle. Although nutrition is the major factor influencing the fatty acid composition of ruminant-derived foods, further progress can be expected through the use of genomic or marker-assisted selection to increase the frequency of favourable genotypes and the formulation of diets to exploit this genetic potential.

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Two novel COLVI long chains in zebrafish that are essential for muscle development.

PubMed

Ramanoudjame, Laetitia; Rocancourt, Claire; Lainé, Jeanne; Klein, Arnaud; Joassard, Lucette; Gartioux, Corine; Fleury, Marjory; Lyphout, Laura; Kabashi, Edor; Ciura, Sorana; Cousin, Xavier; Allamand, Valérie

2015-12-01

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

PubMed

Leal, Mariana Ferreira; Astur, Diego Costa; Debieux, Pedro; Arliani, Gustavo Gonçalves; Silveira Franciozi, Carlos Eduardo; Loyola, Leonor Casilla; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

2015-01-01

The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

Carbamate-linked cationic lipids with different hydrocarbon chains for gene delivery.

PubMed

Shi, Jia; Yu, Shijun; Zhu, Jie; Zhi, Defu; Zhao, Yinan; Cui, Shaohui; Zhang, Shubiao

2016-05-01

A series of carbamate-linked cationic lipids containing saturated or unsaturated hydrocarbon chains and quaternary ammonium head were designed and synthesized. After recrystallization, carbamate-linked cationic lipids with high purity (over 95%) were obtained. The structures of these lipids were proved by IR spectrum, HR-ESI-MS, HPLC, (1)H NMR and (13)C NMR. The liposomes were prepared by using these cationic lipids and neutral lipid DOPE. Particle size and zeta-potential were studied to show that they were suitable for gene transfection. The DNA-bonding ability of C12:0, C14:0 and C18:1 cationic liposomes was much better than others. The results of transfection showed that hydrophobic chains of these lipids have great effects on their transfection activity. The lipids bearing C12:0, C14:0 saturated chains or C18:1 unsaturated chain showed relatively higher transfection efficiency and lower cytotoxicity. So these cationic lipids could be used as non-viral gene carriers for further studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Domain-swapped T cell receptors improve the safety of TCR gene therapy

PubMed Central

Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

2016-01-01

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

PubMed

Yano, Kenji; Aya, Koichiro; Hirano, Ko; Ordonio, Reynante Lacsamana; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2015-02-01

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells. © 2015 American Society of Plant Biologists. All Rights Reserved.

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK.

PubMed

Machado Benelli, Elaine; Buck, Martin; Polikarpov, Igor; Maltempi de Souza, Emanuel; Cruz, Leonardo M; Pedrosa, Fábio O

2002-07-01

PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

PubMed

Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

2013-04-01

Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Alterations in TNF- and IL-related gene expression in space-flown WI38 human fibroblasts

NASA Technical Reports Server (NTRS)

Semov, Alexandre; Semova, Nathalia; Lacelle, Chantale; Marcotte, Richard; Petroulakis, Emmanuel; Proestou, Gregory; Wang, Eugenia

2002-01-01

Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long-term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor- (TNF) or interleukin- (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS-93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor-associated proteins, NSMAF and PTPN13; three TNF-inducible genes, ABC50, PTX3, and SCYA13; TNF-alpha converting enzyme, IL-1 receptor antagonist, and IL-15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.

LEFPS1, a Tomato Farnesyl Pyrophosphate Gene Highly Expressed during Early Fruit Development1

PubMed Central

Gaffe, Joel; Bru, Jean-Philippe; Causse, Mathilde; Vidal, Alain; Stamitti-Bert, Linda; Carde, Jean-Pierre; Gallusci, Philippe

2000-01-01

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement. PMID:10938353

Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

PubMed

Li, Lingxiao; Wang, Tao; Sun, Yi; Cheng, Gang; Yang, Hui; Wei, Zhiguo; Wang, Ping; Hu, Xiaoxiang; Ren, Liming; Meng, Qingyong; Zhang, Ran; Guo, Ying; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

2012-10-15

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single μ gene was identified, the α gene was not identified in this species.