Physiological Genomics
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Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.
Physiological Genomics - Tập 37 Số 1 - Trang 23-34 - 2009
Full-length cDNAs: more than just reaching the ends The development of functional genomic resources is essential to understand and utilize information generated from genome sequencing projects. Central to the development of this technology is the creation of high-quality cDNA resources and improved technologies for analyzing coding and noncoding mRNA sequences. The isolation and mapping of cDNAs is an entrée to characterizing the information that is of significant biological relevance in the genome of an organism. However, a bottleneck is often encountered when attempting to bring to full-length (or at least full-coding) a number of incomplete cDNAs in parallel, since this involves the nonsystematic, time consuming, and labor-intensive iterative screening of a number of cDNA libraries of variable quality and/or directed strategies to process individual clones (e.g., 5′ rapid amplification of cDNA ends). Here, we review the current state of the art in cDNA library generation, as well as present an analysis of the different steps involved in cDNA library generation.
Physiological Genomics - Tập 6 Số 2 - Trang 57-80 - 2001
Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects The vacuolated lens ( vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract, and occasionally a white belly spot. We previously reported that 1) the vl phenotypes are due to a mutation in an orphan G protein-coupled receptor (GPCR), Gpr161; 2) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked quantitative trait loci (QTL) to be mapped (modifiers of vacuolated lens, Modvl1-3); and 3) phenotype-based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract-modifying effect of Modvl3. We now extend this analysis in three ways. First, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined that ∼50% of vl/vl NTD-affected embryos die during development. Second, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed, and two novel modifiers were mapped [ Modvl4, logarithm of odds ratio (LOD) 4.4; Modvl5, LOD 5.0]. Third, phenotype-based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects ( Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were resequenced and nonsynonymous coding variants were identified. Bioinformatics and protein modeling suggest that these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.
Physiological Genomics - Tập 35 Số 3 - Trang 296-304 - 2008
Pathogenic peptide deviations support a model of adaptive evolution of chordate cardiac performance by troponin mutations In cardiac muscle, the troponin (cTn) complex is a key regulator of myofilament calcium sensitivity because it serves as a molecular switch required for translating myocyte calcium fluxes into sarcomeric contraction and relaxation. Studies of several species suggest that ectotherm chordates have myofilaments with heightened calcium responsiveness. However, genetic polymorphisms in cTn that cause increased myofilament sensitivity to activating calcium in mammals result in cardiac disease including arrhythmias, diastolic dysfunction, and increased susceptibility to sudden cardiac death. We hypothesized that specific residue modifications in the regulatory arm of troponin I (TnI) were critical in mediating the observed decrease in myofilament calcium sensitivity within the mammalian taxa. We performed large-scale phylogenetic analysis, atomic resolution molecular dynamics simulations and modeling, and computational alanine scanning. This study provides evidence that a His to Ala substitution within mammalian cardiac TnI (cTnI) reduced the thermodynamic potential at the interface between cTnI and cardiac TnC (cTnC) in the calcium-saturated state by disrupting a strong intermolecular electrostatic interaction. This key residue modification reduced myofilament calcium sensitivity by making cTnI molecularly untethered from cTnC. To meet the requirements for refined mammalian adult cardiac performance, we propose that compensatory evolutionary pressures favored mutations that enhanced the relaxation properties of cTn by decreasing its sensitivity to activating calcium.
Physiological Genomics - Tập 42 Số 2 - Trang 287-299 - 2010
Evolution of the regulatory control of vertebrate striated muscle: the roles of troponin I and myosin binding protein-C Troponin I (TnI) and myosin binding protein-C (MyBP-C) are key regulatory proteins of contractile function in vertebrate muscle. TnI modulates the Ca2+ activation signal, while MyBP-C regulates cross-bridge cycling kinetics. In vertebrates, each protein is distributed as tissue-specific paralogs in fast skeletal (fs), slow skeletal (ss), and cardiac (c) muscles. The purpose of this study is to characterize how TnI and MyBP-C have changed during the evolution of vertebrate striated muscle and how tissue-specific paralogs have adapted to different physiological conditions. To accomplish this we have completed phylogenetic analyses using the amino acid sequences of all known TnI and MyBP-C isoforms. This includes 99 TnI sequences (fs, ss, and c) from 51 different species and 62 MyBP-C sequences from 26 species, with representatives from each vertebrate group. Results indicate that the role of protein kinase A (PKA) and protein kinase C (PKC) in regulating contractile function has changed during the evolution of vertebrate striated muscle. This is reflected in an increased number of phosphorylatable sites in cTnI and cMyBP-C in endothermic vertebrates and the loss of two PKC sites in fsTnI in a common ancestor of mammals, birds, and reptiles. In addition, we find that His132 , Val134 , and Asn141 in human ssTnI, previously identified as enabling contractile function during cellular acidosis, are present in all vertebrate cTnI isoforms except those from monotremes, marsupials, and eutherian mammals. This suggests that the replacement of these residues with alternative residues coincides with the evolution of endothermy in the mammalian lineage.
Physiological Genomics - Tập 42 Số 3 - Trang 406-419 - 2010
Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. Conclusion: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis.
Physiological Genomics - Tập 37 Số 3 - Trang 187-198 - 2009
Cell cycle arrest in a model of colistin nephrotoxicity Colistin (polymixin E) is an antibiotic prescribed with resurging frequency for multidrug resistant gram negative bacterial infections. It is associated with nephrotoxicity in humans in up to 55% of cases. Little is known regarding genes involved in colistin nephrotoxicity. A murine model of colistin-mediated kidney injury was developed. C57/BL6 mice were administered saline or colistin at a dose of 16 mg/kg/day in 2 divided intraperitoneal doses and killed after either 3 or 15 days of colistin. After 15 days, mice exposed to colistin had elevated blood urea nitrogen (BUN), creatinine, and pathologic evidence of acute tubular necrosis and apoptosis. After 3 days, mice had neither BUN elevation nor substantial pathologic injury; however, urinary neutrophil gelatinase-associated lipocalin was elevated ( P = 0.017). An Illumina gene expression array was performed on kidney RNA harvested 72 h after first colistin dose to identify differentially expressed genes early in drug treatment. Array data revealed 21 differentially expressed genes (false discovery rate < 0.1) between control and colistin-exposed mice, including LGALS3 and CCNB1. The gene signature was significantly enriched for genes involved in cell cycle proliferation. RT-PCR, immunoblot, and immunostaining validated the relevance of key genes and proteins. This murine model offers insights into the potential mechanism of colistin-mediated nephrotoxicity. Further studies will determine whether the identified genes play a causative or protective role in colistin-induced nephrotoxicity.
Physiological Genomics - Tập 45 Số 19 - Trang 877-888 - 2013
From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite ( Colinus virginianus ) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT.
Physiological Genomics - Tập 42 Số 2 - Trang 219-235 - 2010
The<i>sld</i>mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression NFS/N- sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187–191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in “atypical” exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. “Atypical” cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.
Physiological Genomics - Tập 14 Số 2 - Trang 95-106 - 2003
Microarray analysis of Akt1 activation in transgenic mouse hearts reveals transcript expression profiles associated with compensatory hypertrophy and failure To investigate molecular mechanisms involved in the development of cardiac hypertrophy and heart failure, we developed a tetracycline-regulated transgenic system to conditionally switch a constitutively active form of the Akt1 protein kinase on or off in the adult heart. Short-term activation (2 wk) of Akt1 resulted in completely reversible hypertrophy with maintained contractility. In contrast, chronic Akt1 activation (6 wk) induced extensive cardiac hypertrophy, severe contractile dysfunction, and massive interstitial fibrosis. The focus of this study was to create a transcript expression profile of the heart as it undergoes reversible Akt1-mediated hypertrophy and during the transition from compensated hypertrophy to heart failure. Heart tissue was analyzed before transgene induction, 2 wk after transgene induction, 2 wk of transgene induction followed by 2 days of repression, 6 wk after transgene induction, and 6 wk of transgene induction followed by 2 wk of repression. Acute overexpression of Akt1 (2 wk) leads to changes in the expression of 826 transcripts relative to noninduced hearts, whereas chronic induction (6 wk) led to changes in the expression of 1,611, of which 65% represented transcripts that were regulated during the pathological phase of heart growth. Another set of genes identified was uniquely regulated during heart regression but not growth, indicating that nonoverlapping transcription programs participate in the processes of cardiac hypertrophy and atrophy. These data define the gene regulatory programs downstream of Akt that control heart size and contribute to the transition from compensatory hypertrophy to heart failure.
Physiological Genomics - Tập 27 Số 2 - Trang 156-170 - 2006
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