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Bioinformatic Analysis (bioinformatic + analysis)
Selected AbstractsProteomic analysis of osteogenic differentiation of dental follicle precursor cellsELECTROPHORESIS, Issue 7 2009Christian Morsczeck Abstract Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs. [source] Molecular characterization of the G,-globin-Tag transgenic mouse model of hormone refractory prostate cancer: Comparison to human prostate cancer,THE PROSTATE, Issue 6 2010Alfonso Calvo Abstract BACKGROUND Prostate cancer (PrCa) has a high incidence in Western countries and at present, there is no cure for hormone refractory prostate cancer. Transgenic mouse models have proven useful for understanding mechanisms of prostate carcinogenesis. The characterization of genetically modified mouse PrCa models using high-throughput genomic analyses provides important information to guide appropriate experiment applications for such model. METHODS We have analyzed the transcriptome of the hormone refractory and highly metastatic Fetal Globin-SV40/T-antigen (G,-globin-Tag) transgenic mouse model for PrCa compared to normal mouse prostate tissue. Gene expression patterns found in G,-globin-Tag mouse prostate tumors were compared with publicly available human localized and metastatic prostate tumors (GEO accession # GSE3325) through hierarchical cluster analysis, Pearson's rank correlation coefficient, and Self Organizing Feature Maps (SOM) analyses. RESULTS G,-globin-Tag tumors clustered closely with human metastatic tumors and gene expression patterns had a significant correlation (P,<,0.01), unlike human localized primary tumors (P,>,0.6). Bioinformatic analyses identified deregulated genetic pathways and networks in G,-globin-Tag tumors, which displayed similarities to alterations in human PrCa. Changes in the expression of genes involved in DNA replication and repair (Rb1, p53, Myc, PCNA, DNMT3A) and growth factor signaling pathways (TGF,2, ERK1/2, NRas, and Notch1) are deregulated in the G,-globin-Tag tumors, suggesting their key role in the oncogenic process. Identification of an enrichment of putative binding sites for transcription factors revealed eight transcription factors that may be important in G,-globin-Tag carcinogenesis, including SP1, NF-Y, CREB, Elk1, and E2F. Novel genes related to microtubule regulation were also identified in G,-globin-Tag tumors as potentially important candidate targets for PrCa. Overexpression of stathmin-1, whose expression was increased in human metastatic prostate tumors, was validated in G,-globin-Tag tumors by immunohistochemistry. This protein belongs to the SV40/T-antigen cancer signature identified in previous studies in prostate, breast, and lung cancer mouse models. CONCLUSIONS Our results show that the G,-globin-Tag model for hormone refractory PrCa shares important features with aggressive, metastatic human PrCa. Given the role of stathmin-1 in the destabilization of microtubles and taxane resistance, the G,-globin-Tag model and other SV40/T-antigen driven transgenic models may be useful for testing potential therapies directed at stathmin-1 in human prostate tumors. Prostate 70: 630,645, 2010. Published 2010 Wiley-Liss, Inc. [source] Identification of genes encoding N -glycan processing ,- N -acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systemsBIOTECHNOLOGY PROGRESS, Issue 1 2010Christoph Geisler Abstract Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N -glycans in place of the complex N -glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N -glycan processing ,- N -acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N -glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing ,- N -acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing ,- N -acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing ,- N -acetylglucosaminidases than degradative or chitinolytic ,- N -acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing ,- N -acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Long-term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk-1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006Antje Lindecke Abstract Long-term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5-dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7000 probes. The DHPG-induced expression of immediate early genes (c-fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk-1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG-dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c-fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk-1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk-1-regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression. [source] Gene expression and dental enamel structure in developing mouse incisorEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2010Amer Sehic Sehic A, Risnes S, Khan Q-E-S, Khuu C, Osmundsen H. Gene expression and dental enamel structure in developing mouse incisor. Eur J Oral Sci 2010; 118: 118,130. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription,polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions. [source] Splicing site mutations in dentin sialophosphoprotein causing dentinogenesis imperfecta type IIEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 5 2006Heidi Holappa Dentinogenesis imperfecta (DGI) type II (OMIM # 125490) is an inherited disorder affecting dentin. Defective dentin formation results in discolored teeth that are prone to attrition and fracture. To date, several mutations have been described in the dentin sialophosphoprotein (DSPP) gene, causing DGI types II and III and dentin dysplasia type II. DSPP encodes two proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here, we describe a mutational analysis of DSPP in seven Finnish families with DGI type II. We report two mutations and five single nucleotide polymorphisms. In one family we found a mutation that has been described earlier in families with different ethnicity, while in six families we found a novel g.1194C>A (IVS2-3) transversion. Bioinformatic analysis of known DSPP mutations suggests that DGI type II is usually caused by aberration of normal splicing. [source] Mutagenic probes of the role of Ser209 on the cavity shaping loop of human monoamine oxidase AFEBS JOURNAL, Issue 16 2009Jin Wang The available literature implicating human monoamine oxidase A (MAO A) in apoptotic processes reports levels of MAO A protein that do not correlate with activity, suggesting that unknown mechanisms may be involved in the regulation of catalytic function. Bioinformatic analysis suggests Ser209 as a possible phosphorylation site that may be relevant to catalytic function because it is adjacent to a six-residue loop termed the ,cavity shaping loop' from structural data. To probe the functional role of this site, MAO A Ser209Ala and Ser209Glu mutants were created and investigated. In its membrane-bound form, the MAO A Ser209Glu phosphorylation mimic exhibits catalytic and inhibitor binding properties similar to those of wild-type MAO A. Solubilization in detergent solution and purification of the Ser209Glu mutant results in considerable decreases in these functional parameters. By contrast, the MAO A Ser209Ala mutant exhibits similar catalytic properties to those of wild-type enzyme when purified. Compared to purified wild-type and Ser209Ala MAO A proteins, the Ser209Glu MAO A mutant shows significant differences in covalent flavin fluorescence yield, CD spectra and thermal stability. These structural differences in the purified MAO A Ser209Glu mutant are not exhibited in quantitative structure,activity relationship patterns using a series of para -substituted benzylamine analogs similar to the wild-type enzyme. These data suggest that Ser209 in MAO A does not appear to be the putative phosphorylation site for regulation of MAO A activity and demonstrate that the membrane environment plays a significant role in stabilizing the structure of MAO A and its mutant forms. [source] Identification of genetic determinants of IGF-1 levels and longevity among mouse inbred strainsAGING CELL, Issue 5 2010Magalie S. Leduc Summary The IGF-1 signaling pathway plays an important role in regulating longevity. To identify the genetic loci and genes that regulate plasma IGF-1 levels, we intercrossed MRL/MpJ and SM/J, inbred mouse strains that differ in IGF-1 levels. Quantitative trait loci (QTL) analysis of IGF-1 levels of these F2 mice detected four QTL on chromosomes (Chrs) 9 (48 Mb), 10 (86 Mb), 15 (18 Mb), and 17 (85 Mb). Haplotype association mapping of IGF-1 levels in 28 domesticated inbred strains identified three suggestive loci in females on Chrs 2 (13 Mb), 10 (88 Mb), and 17 (28 Mb) and in four males on Chrs 1 (159 Mb), 3 (52 and 58 Mb), and 16 (74 Mb). Except for the QTL on Chr 9 and 16, all loci co-localized with IGF-1 QTL previously identified in other mouse crosses. The most significant locus was the QTL on Chr 10, which contains the Igf1 gene and which had a LOD score of 31.8. Haplotype analysis among 28 domesticated inbred strains revealed a major QTL on Chr 10 overlapping with the QTL identified in the F2 mice. This locus showed three major haplotypes; strains with haplotype 1 had significantly lower plasma IGF-1 and extended longevity (P < 0.05) than strains with haplotype 2 or 3. Bioinformatic analysis, combined with sequencing and expression studies, showed that Igf1 is the most likely QTL gene, but that other genes may also play a role in this strong QTL. [source] A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional ComplementationJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2006Zhi-Hua Liao Abstract Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a rate-limiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pI of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homology-based structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant. This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level. (Managing editor: Wei Wang) [source] Bioinformatic analysis of the hepadnavirus e-antigen and its precursor identifies remarkable sequence conservation in all orthohepadnavirusesJOURNAL OF MEDICAL VIROLOGY, Issue 1 2010Peter Revill Abstract The hepatitis B e-antigen (HBeAg) is a non-particulate secretory protein expressed by all viruses within the family Hepadnaviridae. It is not essential for viral assembly or replication but is important for establishment of persistent infection in vivo. Although the exact mechanism(s) by which the HBeAg manifests chronicity are unclear, the HBeAg elicits both humoral and cell-mediated immunity, down-regulates the innate immune response to infection, as well as functioning as a T cell tolerogen and regulating the immune response to the intracellular nucleocapsid. A bioinformatics approach was used to show that the HBeAg and precursory genetic codes share remarkable sequence conservation in all mammalian-infecting hepadnaviruses, irrespective of host, genotype, or geographic origin. Whilst much of this sequence conservation was within key immunomodulatory epitopes, highest conservation was observed at the unique HBeAg N-terminus, suggesting this sequence in particular may play an important role in HBeAg function. J. Med. Virol. 82:104,115, 2010. © 2009 Wiley-Liss, Inc. [source] Differential gene expression in LPS/IFN, activated microglia and macrophages: in vitro versus in vivoJOURNAL OF NEUROCHEMISTRY, Issue 2009Christoph D. Schmid Abstract Two different macrophage populations contribute to CNS neuroinflammation: CNS-resident microglia and CNS-infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN),-treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2-fold to 250-fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFN,-injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the ,microglial' molecules (such as the tolerance associated transcript, Tmem176b) in CNS-infiltrating macrophages. Bioinformatic analysis of the ,19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFN,-activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS-infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS-resident microglia are heterogeneous and thus a universal microglia-specific marker may not exist; (ii) the CNS micro-environment plays significant roles in determining the phenotypes of both CNS-resident microglia and CNS-infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti-inflammatory molecules. [source] Proteomic profiling of KATP channel-deficient hypertensive heart maps risk for maladaptive cardiomyopathic outcomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009Jelena Zlatkovic Abstract KCNJ11 null mutants, lacking Kir6.2 ATP-sensitive K+ (KATP) channels, exhibit a marked susceptibility towards hypertension (HTN)-induced heart failure. To gain insight into the molecular alterations induced by knockout of this metabolic sensor under hemodynamic stress, wild-type (WT) and Kir6.2 knockout (Kir6.2-KO) cardiac proteomes were profiled by comparative 2-DE and Orbitrap MS. Despite equivalent systemic HTN produced by chronic hyperaldosteronism, 114 unique proteins were altered in Kir6.2-KO compared to WT hearts. Bioinformatic analysis linked the primary biological function of the KATP channel-dependent protein cohort to energetic metabolism (64% of proteins), followed by signaling infrastructure (36%) including oxidoreductases, stress-related chaperones, processes supporting protein degradation, transcription and translation, and cytostructure. Mapped protein,protein relationships authenticated the primary impact on metabolic pathways, delineating the KATP channel-dependent subproteome within a nonstochastic network. Iterative systems interrogation of the proteomic web prioritized heart-specific adverse effects, i.e., "Cardiac Damage", "Cardiac Enlargement", and "Cardiac Fibrosis", exposing a predisposition for the development of cardiomyopathic traits in the hypertensive Kir6.2-KO. Validating this maladaptive forecast, phenotyping documented an aggravated myocardial contractile performance, a massive interstitial fibrosis and an exaggerated left ventricular size, all prognostic indices of poor outcome. Thus, Kir6.2 ablation engenders unfavorable proteomic remodeling in hypertensive hearts, providing a composite molecular substrate for pathologic stress-associated cardiovascular disease. [source] Genome-wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a ,phoP mutantPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Antonio Rodríguez-García Abstract Phosphate limitation in Streptomyces and in other bacteria triggers expression changes of a large number of genes. This response is mediated by the two-component PhoR,PhoP system. A Streptomyces coelicolor ,phoP mutant (lacking phoP) has been obtained by gene replacement. A genome-wide analysis of the primary response to phosphate limitation using transcriptomic and proteomic studies has been made in the parental S. coelicolor M145 and in the ,phoP mutant strains. Statistical analysis of the contrasts between the four sets of data generated (two strains under two phosphate conditions) allowed the classification of all genes into 12 types of profiles. The primary response to phosphate limitation involves upregulation of genes encoding scavenging enzymes needed to obtain phosphate from different phosphorylated organic compounds and overexpression of the high-affinity phosphate transport system pstSCAB. Clear interactions have been found between phosphate metabolism and expression of nitrogen-regulated genes and between phosphate and nitrate respiration genes. PhoP-dependent repressions of antibiotic biosynthesis and of the morphological differentiation genes correlated with the observed ,phoP mutant phenotype. Bioinformatic analysis of the presence of PHO boxes (PhoP-binding sequences) in the upstream regions of PhoP-controlled genes were validated by binding of PhoP, as shown by electrophoretic mobility shift assays. [source] Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expressionARTHRITIS & RHEUMATISM, Issue 8 2009Laura M. Elsby Objective Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator involved in the pathogenesis of rheumatoid arthritis. This study was undertaken to identify the MIF promoter elements responsible for regulating gene expression. Methods Luciferase reporter gene assays were used to identify the MIF promoter sequence responsible for basal activity. Bioinformatic analysis was used to predict transcription factor binding sites, and electrophoretic mobility shift assay (EMSA) was used to demonstrate transcription factor binding. Chromatin immunoprecipitation (ChIP) was used to demonstrate transcription factor loading on the MIF promoter. Results We identified the minimal promoter sequence required for basal MIF promoter activity that was also capable of conferring glucocorticoid-dependent inhibition in a T lymphocyte model cell line. Deletion studies and EMSA revealed 2 elements in the MIF promoter that were responsible for basal promoter activity. The 5, element binds CREB/activating transcription factor 1, and the 3, element is a functional hypoxia-responsive element binding hypoxia-inducible factor 1,. Further studies demonstrated that the cis elements are both required for glucocorticoid-dependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor , to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3. Conclusion Our findings indicate that hypoxia and glucocorticoid signaling converge on a single element regulating MIF; this regulatory unit is a potential interacting node for microenvironment sensing of oxygen tension and glucocorticoid action in foci of inflammation. [source] Upstream genetic variant near INSIG2, influences response to carnitine supplementation in bipolar patients with valproate-induced weight gainACTA NEUROPSYCHIATRICA, Issue 3 2009K Doudney Background: The protein product of INSIG2 is involved in cholesterol and triglyceride metabolism and homeostasis. Variation at rs7566605 near the gene INSIG2 has been associated with increased BMI. Objective: To evaluate the effect of rs7566605/INSIG2 genotype on the ability of valproate-treated bipolar patients (BMI , 25 kg/m2) to lose weight using carnitine supplementation during a 26-week lifestyle intervention study. Design: Forty-eight bipolar patients with clinically significant treatment emergent weight gain were genotyped at the rs7566605 SNP. Participants were randomised to l -carnitine (15 mg/kg/day) or placebo for 26 weeks in conjunction with a moderately energy restricted, low-fat diet. Weight and body fat percent were measured fortnightly. Waist circumference measurements and dual-energy X-ray absorptiometry were used to assess changes in body composition. Obesity-related biomarkers were measured at baseline and 26 weeks. Results: There was a significant interaction between rs7566605/INSIG2 genetic status and treatment with carnitine or placebo. Carnitine had no significant effect on body composition measures in G allele homozygous patients who lost between 0.97 and 2.23 kg of fat. However C allele carriers on average gained 2.28 kg when given a placebo. Carnitine supplementation in this group enabled average weight loss of 2.22 kg of fat (p = 0.01). Approximately half of this mass was in the vital truncal compartment (p = 0.002). Bioinformatic analysis detected that the SNP lies in a highly conserved 336 bp sequence which potentially affects INSIG2 gene expression. Conclusions: C-carriers at rs7566605, possibly regulating the homeostasis gene INSIG2, lost significantly less weight in this lifestyle intervention study. This effect was reversed by carnitine supplementation. [source] Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regenerationDEVELOPMENTAL DYNAMICS, Issue 10 2006Matthew Grow Abstract Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues. Developmental Dynamics 235:2667,2685, 2006. © 2006 Wiley-Liss, Inc. [source] Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisumINSECT MOLECULAR BIOLOGY, Issue 2010J. Huybrechts Abstract Aphids respond to environmental changes by developing alternative phenotypes with differing reproductive modes. Parthenogenetic reproduction occurs in spring and summer, whereas decreasing day lengths in autumn provoke the production of sexual forms. Changing environmental signals are relayed by brain neuroendocrine signals to the ovarioles. We combined bioinformatic analyses with brain peptidomics and cDNA analyses to establish a catalogue of pea aphid neuropeptides and neurohormones. 42 genes encoding neuropeptides and neurohormones were identified, of which several were supported by expressed sequence tags and/or peptide mass analyses. Interesting features of the pea aphid peptidome are the absence of genes coding for corazonin, vasopressin and sulfakinin and the presence of 10 different genes coding insulin related peptides, one of which appears to be very abundantly expressed. [source] Deciphering the human nucleolar proteome,MASS SPECTROMETRY REVIEWS, Issue 2 2006Yohann Couté Abstract Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states. © 2005 Wiley Periodicals, Inc. Mass Spec Rev 25:215,234, 2006 [source] Gene Expression Profiling of Nasal Polyps Associated With Chronic Sinusitis and Aspirin-Sensitive Asthma,THE LARYNGOSCOPE, Issue 5 2008Konstantina M. Stankovic MD Abstract Objective: To identify genes whose expression is most characteristic of chronic rhinosinusitis and aspirin-sensitive asthma through genome-wide transcriptional profiling of nasal polyp tissue. Study Design: Prospective, controlled study conducted at a tertiary care institution. Methods: Thirty genome-wide expression microarrays were used to compare nasal polyp tissue from patients with chronic rhinosinusitis alone (CRS, n = 10) or chronic rhinosinusitis and a history of aspirin-sensitive asthma (ASA, n = 10) to normal sinonasal mucosa from patients who underwent surgery for non-sinus related conditions (controls, n = 10). Genes found to be most characteristic of each polyp phenotype, as determined from bioinformatic analyses, were validated using real-time quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry in different patient sets. Results: The transcriptional signature of the control mucosa was distinctly different from that of either polyp phenotype. Genes most characteristic of the CRS phenotype included two upregulated genes,met proto-oncogene (MET) and protein phosphatase 1 regulatory subunit 9B (PPP1R9B),and two downregulated genes, prolactin-induced protein (PIP) and zinc alpha2-glycoprotein (AZGP1). The gene most characteristic of the ASA phenotype was periostin (POSTN), which was upregulated relative to controls. Differences between the CRS and ASA phenotypes were associated with alterations in the 6p22, 22q13, and 1q23 chromosomal regions. Conclusions: Nasal polyps appear to have characteristic transcriptional signatures compared to normal sinonasal mucosa. The five genes identified in this study likely play roles in the pathogenesis of polyps associated with CRS and ASA, and are therefore attractive targets for novel medical therapies for these common debilitating diseases. [source] Definition and spatial annotation of the dynamic secretome during early kidney developmentDEVELOPMENTAL DYNAMICS, Issue 6 2006Gemma Martinez Abstract The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350,1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants. Developmental Dynamics 235:1709,1719, 2006. © 2006 Wiley-Liss, Inc. [source] Gene expression and dental enamel structure in developing mouse incisorEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2010Amer Sehic Sehic A, Risnes S, Khan Q-E-S, Khuu C, Osmundsen H. Gene expression and dental enamel structure in developing mouse incisor. Eur J Oral Sci 2010; 118: 118,130. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription,polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions. [source] Functional profiling of uncommon VCAM1 promoter polymorphisms prevalent in African American populations,,HUMAN MUTATION, Issue 8 2007Gila Idelman Abstract Multiple variants of the vascular adhesion molecule-1 (VCAM1) promoter show increased nucleotide heterozygosity in the African American population. Using a novel transfection-based transcriptional pathway profiling method, we show that select uncommon variants are functionally hyperactive. Eight candidate VCAM1 promoter haplotypes comprising 13 previously identified SNPs were assessed for response to known mitogens. Activity was correlated with bioinformatic analysis of hyper- and hyporesponsive variants to identify the gain or loss of haplotype-specific transcription factor binding site (TFBS). Using this approach, a low frequency regulatory allele (c.,540A>G; dbSNP rs3783605:A>G), found in a hyperactive VCAM1 promoter haplotype, was shown to create a candidate binding site for ETS2 that was confirmed in vivo by chromatin immunoprecipitation. This report provides the first functional evaluation of VCAM1 promoter polymorphisms and establishes a hypothetical foundation for investigation of their role in the pathogenesis of VCAM1 -associated diseases that disproportionately afflict African Americans, including thromboembolic diseases, asthma, and multiple myeloma. Hum Mutat 28(8), 824,829, 2007. Published 2007, Wiley-Liss, Inc. [source] Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy,HUMAN MUTATION, Issue 2 2007Christophe Béroud Abstract Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients. Hum Mutat 28(2), 196,202, 2007. © 2006 Wiley-Liss, Inc. [source] Molecular characterization of two novel milk proteins in the tsetse fly (Glossina morsitans morsitans)INSECT MOLECULAR BIOLOGY, Issue 2 2010G. Yang Abstract Purpose: Milk proteins are an essential component of viviparous reproduction in the tsetse fly. Milk proteins are synthesized in and secreted from the milk gland tissue and constitute 50% of the secretions from which the intrauterine larva derives its nourishment. To understand milk protein function and regulation during viviparous reproduction, milk proteins need to be identified and characterized. Methods: Two putative unknown secretory proteins (GmmMGP2 and GmmMGP3) were selected by bioinformatic analysis of tissue specific tsetse cDNA libraries. RT-PCR analysis was performed to verify their milk gland/fat body specific expression profile. Detailed characterization of developmental and tissue specific expression of these proteins was performed by northern blot analysis and fluorescent in situ hybridization. Functional analysis of the milk gland proteins during the tsetse gonotrophic cycle was performed using RNA interference (RNAi). Results: The predicted proteins from gmmmgp2 and gmmmgp3 are small ,22 kD and contain a high proportion of hydrophobic amino acids and potential phosphorylation sites. Expression of both genes is tissue specific to the secretory cells of the milk gland. Transcript abundance for both genes increases over the course of intrauterine larval development and parallels that of gmmmgp, a well characterized milk protein gene considered to be the major milk protein. Phenotypic analysis of flies after RNA interference treatment revealed a significant effect upon fecundity in the gmmmgp2 knockdown flies, but not the gmmmgp3 flies. Knockdown of gmmmgp2 resulted in disruption of ovulation and consequent oocyte accumulation and degradation. Gmmmgp2 knockdown also had a significant impact on fly mortality. Conclusions: This work identifies two novel genes, the proteins of which appear to function in response to intrauterine larvigenesis in tsetse. These proteins may be nutritional components of the milk secretions provided to the larva from the mother. Phenotypic data from knockdown of gmmmgp2 suggests that this protein may also have a regulatory function given the defect in ovulation observed in knockdown flies. Further analysis of these genes will be important (in conjunction with other milk proteins) for identification of transcriptional regulation mechanisms that direct milk gland/pregnancy specific gene expression. [source] SINE insertion polymorphism on the X chromosome differentiates Anopheles gambiae molecular formsINSECT MOLECULAR BIOLOGY, Issue 4 2005M. J. Barnes Abstract Polymorphic SINE insertions can be useful markers for assessing population structure and differentiation. Maque is a family of SINE elements which, based on bioinformatic analysis, was suggested to have been active recently in Anopheles gambiae, the major vector of malaria. Here, we report the development of polymorphic Maque insertions as population genetic markers in A. gambiae, and the use of these markers to better characterize divergence on the X chromosome between A. gambiae M and S molecular forms in populations from Burkina Faso and Mali. Our data are consistent with the recent activity of Maque. Phylogenetic analysis suggests that at least two recently active lineages may have a role in mediating genome evolution. We found differences in element insertion frequency and sequence between the M and S populations analysed. Significant differentiation was observed between these two groups across a 6 Mb region at the proximal (centromeric) end of the X chromosome. Locus-specific FST values ranged from 0.14 to 1.00 in this region, yet were not significantly different from zero in more distal locations on the X chromosome; the trend was consistent in populations from both geographical locales suggesting that differentiation is not due to local adaptation. Strong differentiation between M and S at the proximal end of the X chromosome, but not outside this region, suggests the action of selection counteracting limited gene flow between these taxa and supports their characterization as incipient species. [source] Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/,-catenin signaling pathway,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Macarena S. Arrázola Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of ,-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide. J. Cell. Physiol. 221: 658,667, 2009. © 2009 Wiley-Liss, Inc. [source] A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional ComplementationJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2006Zhi-Hua Liao Abstract Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a rate-limiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pI of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homology-based structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant. This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level. (Managing editor: Wei Wang) [source] Biochemical aspects of the neuroprotective mechanism of PTEN-induced kinase-1 (PINK1)JOURNAL OF NEUROCHEMISTRY, Issue 1 2008Ryan D. Mills Abstract Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis. [source] Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient miceMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2010Sanny S.W. Chung Abstract Retinoic acid receptor alpha (RAR,)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell,cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RAR,-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RAR,-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RAR,-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a downregulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stressMOLECULAR ECOLOGY, Issue 11 2009MARGIT EGG Abstract Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains , apart from the regulatory promoter region , several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions. [source] | ||||||||||||||||||||||||||||