EVOLUTION, Issue 4 2005
Michael A. D. Goodisman
Abstract Holometabolous insects inhabit almost every terrestrial ecosystem. The evolutionary success of holometabolous insects stems partly from their developmental program, which includes discrete larval and adult stages. To gain an understanding of how development differs among holometabolous insect taxa, we used cDNA microarray technology to examine differences in gene expression between larval and adult Camponotus festinatus ants. We then compared expression patterns obtained from our study to those observed in the fruitfly Drosophila melanogaster. We found that many genes showed distinct patterns of expression between the larval and adult ant life stages, a result that was confirmed through quantitative reverse-transcriptase polymerase chain reaction. Genes involved in protein metabolism and possessing structural activity tended to be more highly expressed in larval than adult ants. In contrast, genes relatively upregulated in adults possessed a greater diversity of functions and activities. We also discovered that patterns of expression observed for homologous genes in D. melanogaster differed substantially from those observed in C. festinatus. Our results suggest that the specific molecular mechanisms involved in metamorphosis will differ substantially between insect taxa. Systematic investigation of gene expression during development of other taxa will provide additional information on how developmental pathways evolve. [source]

Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fish

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2008
Akimitsu Miyake
The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [source]

Dynamics of 17,-Ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): Absorption, tissue distribution, and hepatic gene expression pattern

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2006
Ann D. Skillman
Abstract 17,-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor , (ER,) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ER, mRNA, and EE2 were measured using enzymelinked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography,mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ER, mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ER,) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive. [source]

Sympathectomy suppresses tumor growth and alters gene-expression profiles in rat tongue cancer

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2009
Bina Raju
Sympathetic nerves are known to affect carcinogenesis. Recently we found that sympathetic denervation decreases the size of rat tongue tumors. To identify genes involved in rat tongue carcinogenesis and to study the effect of sympathetic nerves on these genes, we compared gene-expression profiles in normal rat tongue (control) and in tumor-induced tongues with (SCGx) and without (Sham) bilateral sympathectomy. Significance analysis of microarrays revealed 280 genes (168 up-regulated, 112 down-regulated) that showed at least a twofold differential expression between Sham and SCGx tumors (false discovery rate < 5%). These included genes associated with cell adhesion, signaling, structure, proliferation, metabolism, angiogenesis, development, and immunity. Hierarchical clustering demonstrated that controls and sympathectomized tumors grouped together, while Sham tumors grouped separately. We identified 34 genes, known to be involved in carcinogenesis, that were not differentially expressed between sympathectomized tumors and control tongues, but which showed a significant change in expression in Sham tumors. Microarray results of 12 of these genes were confirmed by quantitative reverse transcription,polymerase chain reaction. In conclusion, sympathectomy significantly altered the gene-expression profile and inhibited tumor growth. The expression of several cancer genes were increased more than threefold in Sham tumors, but unaltered in the sympathectomized tumors when compared with controls, indicating that these genes may be of significance in rat tongue carcinogenesis. [source]

Efficient use of DNA molecular markers to construct industrial yeast strains

FEMS YEAST RESEARCH, Issue 8 2007
Philippe Marullo
Abstract Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50,170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure. [source]

Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridization,,

HUMAN MUTATION, Issue 11 2007
Yao-Shan Fan
Abstract Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459,kb to 19,Mb while revealing a small number of CNVs (0.72/individual). Another platform with ,240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs. Hum Mutat 28(11),1124,1132, 2007. © 2007 Wiley-Liss, Inc. [source]

Activation of an IL-6:STAT3-dependent transcriptome in pediatric-onset inflammatory bowel disease

INFLAMMATORY BOWEL DISEASES, Issue 4 2008
Rebecca Carey MD
Abstract Background: While activation of the IL-6-dependent transcription factor signal transducer and activator of transcription 3 (STAT3) has been implicated in the pathogenesis of inflammatory bowel disease (IBD), a direct effect on mucosal gene expression and inflammation has not been shown. We hypothesized that a proinflammatory IL-6:STAT3-dependent biological network would be up regulated in pediatric-onset IBD patients, and would be associated with the severity of mucosal inflammation. Methods: Patients with pediatric-onset IBD were enrolled at diagnosis and during therapy. Serum cytokine analysis was performed using Bioplex. STAT3 phosphorylation (pSTAT3) in peripheral blood leukocytes (PBLs) was assessed by flow cytometry. Immunohistochemistry of colonic mucosa was used to localize pSTAT3 and STAT3 target genes. Microarray analysis was used to determine RNA expression profiles from colon biopsies. Results: Circulating IL-6 was upregulated in active IBD patients at diagnosis and during therapy. STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3+/CD4+ lymphocytes, and affected colon biopsies of IBD patients. The frequency of pSTAT3+ PB granulocytes and colon epithelial and lamina propria cells was highly correlated with the degree of mucosal inflammation. Microarray and Ingenuity Systems bioinformatics analysis identified IL-6:STAT3-dependent biological networks upregulated in IBD patients which control leukocyte recruitment, HLA expression, angiogenesis, and tissue remodeling. Conclusions: A proinflammatory IL6:STAT3 biologic network is upregulated in active pediatric IBD patients at diagnosis and during therapy. Specific targeting of this network may be effective in reducing mucosal inflammation. (Inflamm Bowel Dis 2007) [source]

Microarray-based survey of a subset of putative olfactory genes in the mosquito Anopheles gambiae

INSECT MOLECULAR BIOLOGY, Issue 6 2005
H. Biessmann
Abstract Female Anopheles gambiae mosquitoes respond to odours emitted from humans in order to find a blood meal, while males are nectar feeders. This complex behaviour is controlled at several levels, but is probably initiated by the interaction of various molecules in the antennal sensilla. Important molecules in the early odour recognition events include odourant binding proteins (OBPs), which may be involved in odour molecule transport, odourant receptors (ORs) that are expressed in the chemosensory neurones and odour degrading enzymes (ODEs). To obtain a better understanding of the expression patterns of genes that may be involved in host odour reception in females, we generated a custom microarray to study their steady state mRNA levels in chemosensory tissues, antennae and palps. These results were supported by quantitative RT PCR. Our study detected several OBPs that are expressed at significantly higher levels in antennae and palps of females vs. males, while others showed the opposite expression pattern. Most OBPs are slightly down-regulated 24 h after blood feeding, but some, especially those with higher expression levels in males, are up-regulated in blood-fed females, suggesting a shift in blood-fed females from human host seeking to nectar feeding. [source]

Tumor-stromal crosstalk in invasion of oral squamous cell carcinoma: a pivotal role of CCL7

INTERNATIONAL JOURNAL OF CANCER, Issue 2 2010
Da-Woon Jung
Abstract Recent studies have shown that stromal fibroblasts have a more profound influence on the initiation and progression of carcinoma than was previously appreciated. This study aimed at investigating the reciprocal relationship between cancer cells and their associated fibroblasts at both the molecular and cellular level in oral squamous cell carcinoma (OSCC). To identify key molecular regulators expressed by carcinoma-associated fibroblasts (CAF) that promote cancer cell invasion, microarrays were performed by comparing cocultured OSCC cells and CAF with monoculture controls. Microarray and real-time PCR analysis identified marked upregulation of the chemokine (C-C motif) ligand 7 (CCL7) in cocultured CAF. ELISA showed an elevated level of CCL7 secretion from CAF stimulated by coculture with OSCC cells. CCL7 promoted the invasion and migration of OSCC cells, and the invasiveness was inhibited by treatment with CCL7 neutralizing antibody. OSCC cells were shown to express CCR1, CCR2 and CCR3, receptors for CCL7, by RT-PCR. In addition, treatment with anti-CCR1 or anti-CCR3 antibody inhibited CCL7-induced OSCC cell migration, implicating that CCL7 promotes cancer cell migration through CCR1 and CCR3 on OSCC cells. Cytokine antibody array analysis of the supernatant from OSCC cell culture revealed that interleukin-1, was an inducer of CCL7 secretion by CAF. This study confirms the reciprocal relationship of the molecular crosstalk regulating the invasion of OSCC and describes new potential targets for future therapy. [source]

Microarray-based DNA profiling to study genomic aberrations,

IUBMB LIFE, Issue 7 2008
Nic Waddell
Abstract High throughput microarrays were initially developed to analyse the expression of many RNA transcripts in parallel. The technology has since been adapted to a variety of applications, one of which is the analysis of the genome to study DNA dosage and sequence content. Advances in microarray fabrication and completion of large-scale genome sequencing projects have enabled the rapid development of affordable array-based methods for high-resolution genome-wide assessment of DNA alterations. This review will describe the evolution of microarray assays to study genomic aberrations and will highlight how they have enabled researchers to gain insight into the biology of human diseases and how they will benefit research in the future. © 2008 IUBMB IUBMB Life, 60(7): 437,440, 2008 [source]

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

JOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006
J.E. Burton
Abstract Aims:, To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. Methods and Results:, Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. Conclusions:, Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. Significance and Impact of the Study:, Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates. [source]

Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004
Diana S de Jong
Abstract Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes. [source]

Adenylyl cyclase-5 activity in the nucleus accumbens regulates anxiety-related behavior

JOURNAL OF NEUROCHEMISTRY, Issue 1 2008
Kyoung-Shim Kim
Abstract Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens (NAc), two brain areas which have been implicated in motor function, reward, and emotion. Here we demonstrate that mice lacking AC5 (AC5,/,) display strong reductions in anxiety-like behavior in several paradigms. This anxiolytic behavior in AC5,/, mice was reduced by the D1 receptor antagonist SCH23390 and enhanced by the D1 dopamine receptor agonist, dihydrexidine (DHX). DHX-stimulated c- fos induction in AC5,/, mice was blunted in the dorso-lateral striatum, but it was overactivated in the dorso-medial striatum and NAc. The siRNA-mediated inhibition of AC5 levels within the NAc was sufficient to produce an anxiolytic-like response. Microarray and RT-PCR analyses revealed an up-regulation of prodynorphin and down-regulation of cholecystokinin (CCK) in the NAc of AC5,/, mice. Administration of nor-binaltorphimine (a kappa opioid receptor antagonist) or CCK-8s (a CCK receptor agonist) reversed the anxiolytic-like behavior exhibited by AC5,/, mutants. Taken together, these results suggest an essential role of AC5 in the NAc for maintaining normal levels of anxiety. [source]

c-DNA Microarray to determine molecular events in neurodegeneration and neuroprotection

JOURNAL OF NEUROCHEMISTRY, Issue 2002
M. B. H. Youdim
Cell death in CNS involves complex processes, many of which have not been identified biochemically. At the present biochemical techniques cannot adequately establish these. However, the advent of cDNA microarray or microchips, in which the expression of thousands of genes can be measured at once to give a global assessment in disease pathology, its progress or animal models, has simplified this. We have employed this technique to study the mechanism of neurotoxicity of MPTP and 6-hydroxydoapmine induced in neuronally derived cells in culture, in the animal models of Parkinson's disease and neuroprotection initiated by monoamine oxidase B inhibitor, rasagiline; iron chelators, R-apomorphine and EGCG and other neuroprotective drugs. Our studies have clearly indicated that MPTP induced early gene expression, prior to cell death (first 24 h), are prerequirement for 51 late gene expression changes implicated at the time of neuronal death. The latter genes include those involved in iron metabolism, oxidative stress, inflammatory processes, glutaminergic excitotoxicity, nitric oxide, growth factors, transcription factors, cell cycle, intermediatory metabolism and other gene previously not identified. The expressions of many of the latter genes, also identified by in situ hybridization, are prevented when the animals are pretreated with the above neuroprotective drugs. These studies have clearly shown that neurodegeneratrion is a complex cascades of ,domino' effect. Thus a single neuroprotective drug treatment may not be adequate to prevent it, but, that a cocktail of drugs might. [source]

Decreased Triadin and Increased Calstabin2 Expression in Great Danes with Dilated Cardiomyopathy

JOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2009
M.A. Oyama
Background: Dilated cardiomyopathy (DCM) is a common cardiac disease of Great Dane dogs, yet very little is known about the underlying molecular abnormalities that contribute to disease. Objective: Discover a set of genes that are differentially expressed in Great Dane dogs with DCM as a way to identify candidate genes for further study as well as to better understand the molecular abnormalities that underlie the disease. Animals: Three Great Dane dogs with end-stage DCM and 3 large breed control dogs. Methods: Prospective study. Transcriptional activity of 42,869 canine DNA sequences was determined with a canine-specific oligonucleotide microarray. Genome expression patterns of left ventricular tissue samples from affected Great Dane dogs were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with expression from large breed dogs with noncardiac disease. Results: Three hundred and twenty-three transcripts were differentially expressed (,2-fold change). The transcript with the greatest degree of upregulation (+61.3-fold) was calstabin2 (FKBP12.6), whereas the transcript with the greatest degree of downregulation (,9.07-fold) was triadin. Calstabin2 and triadin are both regulatory components of the cardiac ryanodine receptor (RyR2) and are critical to normal intracellular Ca2+ release and excitation-contraction coupling. Conclusion and clinical importance: Great Dane dogs with DCM demonstrate abnormal calstabin2 and triadin expression. These changes likely affect Ca2+ flux within cardiac cells and may contribute to the pathophysiology of disease. Microarray-based analysis identifies calstabin2, triadin, and RyR2 function as targets of future study. [source]

A temperature-regulated Campylobacter jejuni gluconate dehydrogenase is involved in respiration-dependent energy conservation and chicken colonization

MOLECULAR MICROBIOLOGY, Issue 2 2008
Mohanasundari Pajaniappan
Summary Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37°C and 42°C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81,176 revealed the upregulation at 42°C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81,176 demonstrated GADH activity, converting d -gluconate to 2-keto- d -gluconate, that was higher at 42°C than at 37°C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d -gluconate or 2-keto- d -gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts. [source]

Microarray-based comparison of genetic differences between strains of Streptomyces turgidiscabies with focus on the pathogenicity island

MOLECULAR PLANT PATHOLOGY, Issue 6 2010
MARJA AITTAMAA
SUMMARY The areas of the pathogenicity island (PAI) designated as ,colonization region' (CR) and ,toxicogenic region' (TR) [Lerat et al. (2009) Mol. Plant Pathol. 10, 579,585] contain genes required for virulence and phytoxin production, respectively, in Streptomyces spp. causing common scab on potatoes. The PAI was tested for genetic variability by microarray analysis in strains of S. turgidiscabies isolated from potatoes in Finland. The data revealed four types of PAI based on divergent CR and TR which occurred in different combinations. Only one PAI type was highly similar to S. scabies (strains 87.22 and ATTC49173). Using probes designed for the predicted genes of S. scabies, two gene clusters in S. scabies appeared to be similar to most strains of S. turgidiscabies and contained PAI genes corresponding to CR and TR. They were located approximately 5 Mb apart in the S. scabies genome, as compared with only 0.3 Mb in S. turgidiscabies Car8. Data from comparative genomic hybridization with probes designed for S. scabies genes and for the PAI of S. turgidiscabies were compared by multilocus cluster analysis, which revealed two strains of S. turgidiscabies that were very closely related at the whole-genome level, but contained distinctly different PAIs. The type strain of S. reticuliscabiei (DSM41804; synonymous to S. turgidiscabies) was clustered with S. turgidiscabies. Taken together, the data indicate wide genetic variability of PAIs among strains of S. turgidiscabies, and demonstrate that PAI is made up of a mosaic of regions which may undergo independent evolution. [source]

Altered expression of mRNA for HIF-1, and its target genes RTP801and VEGF in patients with oral lichen planus

ORAL DISEASES, Issue 3 2010
M Ding
Oral Diseases (2010) 16, 299,304 Objective:, To explore a potential causal contribution of the transcription factor HIF-1, and its target gene, RTP801 and VEGF, to the development of oral lichen planus (OLP). Design relevant:, Twenty-two adult OLP patients were enrolled in this study. All OLP diagnoses were verified by histopathological characteristics. Normal mucous specimens were collected from 12 controls after various oral surgeries. Material and method:, RNA was isolated from OLP and control specimens. Microarray was performed using BiostarH-40s gene chip. Expression of HIF-1,, VEGF and RTP801 was evaluated using quantitative real-time polymerase chain reaction (qPCR). Unpaired t -test and one-way ANOVA was used for statistical analysis. Results:, Microarray results showed that RTP801 expression was lower in OLP than in controls (779 vs 3090). qPCR further confirmed that expression of RTP801 was similarly lower in OLP than in controls (0.363 vs 1.473, P < 0.001); expression of VEGF was also lower in OLP (0.448 vs 1.74, P = 0.012). In contrast, expression of HIF-1, was higher in OLP than in controls (11.12 vs 1.628, P < 0.001). Conclusion:, The oral mucosa of OLP is hypoxic. Genes that are activated by hypoxia, such as RTP801 and VEGF, and their signal cascades may be novel potential therapeutic targets for OLP. [source]

Prenatal diagnosis and molecular cytogenetic analysis of partial monosomy 10q (10q25.3,qter) and partial trisomy 18q (18q23,qter) in a fetus associated with cystic hygroma and ambiguous genitalia

PRENATAL DIAGNOSIS, Issue 6 2005
Chih-Ping Chen
Abstract Objectives To present the prenatal diagnosis and molecular cytogenetic analysis of a fetus with nuchal cystic hygroma and ambiguous genitalia. Case and Methods Amniocentesis was performed at 16 weeks' gestation because of the abnormal fetal sonographic finding of a large septated nuchal cystic hygroma. Genetic amniocentesis revealed a terminal deletion in the long arm of chromosome 10. The paternal karyotype was subsequently found to be 46,XY,t(10;18)(q25.3;q23). The maternal karyotype was normal. The pregnancy was terminated. A hydropic fetus was delivered with a septated nuchal cystic hygroma and ambiguous genitalia. Fluorescence in situ hybridization (FISH), microarray-based comparative genomic hybridization (CGH), and polymorphic DNA markers were used to investigate the involved chromosomal segments. Results FISH study showed absence of the 10q telomeric probe and presence of the 18q telomeric probe in the derivative chromosome 10. Microarray-based CGH analysis showed loss of distal 10q and gain of distal 18q. Polymorphic DNA marker analysis determined the breakpoints. The fetal karyotype was 46,XY,der(10)t(10;18)(q25.3;q23)pat. The chromosome aberration resulted in partial monosomy 10q (10q25.3,qter) and partial trisomy 18q (18q23,qter). Conclusions The present case provides evidence that partial monosomy 10q (10q25.3,qter) with partial trisomy 18q (18q23,qter) can be a genetic cause of fetal cystic hygroma and ambiguous genitalia. Cytogenetic analysis for prenatally detected structural abnormalities may detect unexpected inherited chromosome aberrations. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA Microarray

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010
OM Kandil
Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source]

The Arabidopsis her1 mutant implicates GABA in E -2-hexenal responsiveness

THE PLANT JOURNAL, Issue 2 2008
Rossana Mirabella
Summary When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E -2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E -2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E -2-hexenal-response (her) mutants that showed sustained root growth after E -2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a ,-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E -2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E -2-hexenal, we propose a role for GABA in mediating E -2-hexenal responses. [source]

The Transcriptome of the Implant Biopsy Identifies Donor Kidneys at Increased Risk of Delayed Graft Function

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2008
T. F. Mueller
Improved assessment of donor organ quality at time of transplantation would help in management of potentially usable organs. The transcriptome might correlate with risk of delayed graft function (DGF) better than conventional risk factors. Microarray results of 87 consecutive implantation biopsies taken postreperfusion in 42 deceased (DD) and 45 living (LD) donor kidneys were compared to clinical and histopathology-based scores. Unsupervised analysis separated the 87 kidneys into three groups: LD, DD1 and DD2. Kidneys in DD2 had a greater incidence of DGF (38.1 vs. 9.5%, p < 0.05) than those in DD1. Clinical and histopathological risk scores did not discriminate DD1 from DD2. A total of 1051 transcripts were differentially expressed between DD1 and DD2, but no transcripts separated DGF from immediate graft function (adjusted p < 0.01). Principal components analysis revealed a continuum from LD to DD1 to DD2, i.e. from best to poorest functioning kidneys. Within DD kidneys, the odds ratio for DGF was significantly increased with a transcriptome-based score and recipient age (p < 0.03) but not with clinical or histopathologic scores. The transcriptome reflects kidney quality and susceptibility to DGF better than available clinical and histopathological scoring systems. [source]

Combining Microarray-based Genomic Selection (MGS) with the Illumina Genome Analyzer Platform to Sequence Diploid Target Regions

ANNALS OF HUMAN GENETICS, Issue 5 2009
David T. Okou
Summary Novel methods of targeted sequencing of unique regions from complex eukaryotic genomes have generated a great deal of excitement, but critical demonstrations of these methods efficacy with respect to diploid genotype calling and experimental variation are lacking. To address this issue, we optimized microarray-based genomic selection (MGS) for use with the Illumina Genome Analyzer (IGA). A set of 202 fragments (304 kb total) contained within a 1.7 Mb genomic region on human chromosome X were MGS/IGA sequenced in ten female HapMap samples generating a total of 2.4 GB of DNA sequence. At a minimum coverage threshold of 5X, 93.9% of all bases and 94.9% of segregating sites were called, while 57.7% of bases (57.4% of segregating sites) were called at a 50X threshold. Data accuracy at known segregating sites was 98.9% at 5X coverage, rising to 99.6% at 50X coverage. Accuracy at homozygous sites was 98.7% at 5X sequence coverage and 99.5% at 50X coverage. Although accuracy at heterozygous sites was modestly lower, it was still over 92% at 5X coverage and increased to nearly 97% at 50X coverage. These data provide the first demonstration that MGS/IGA sequencing can generate the very high quality sequence data necessary for human genetics research. All sequences generated in this study have been deposited in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra, Accession # SRA007913). [source]

Microarray: an instrument for cancer surgeons of the future?

ANZ JOURNAL OF SURGERY, Issue 7-8 2010
Matthew L. Broadhead
Abstract Microarray enables the study of thousands of genes simultaneously. While still in its infancy as a technique and with a number of barriers to be overcome, microarray is allowing scientists to thoroughly examine the molecular pathways of cancer pathogenesis. However, the adoption of microarray as a clinically applicable technique has been slow coming. Current literature suggests roles in the diagnosis of tumours of unknown origin, in the evaluation of prognostic markers, and in guiding treatment for recurrent and resistant malignancy. This review outlines the science of microarray and draws on clinical examples, including osteosarcoma, breast, prostate and pancreatic carcinomas, to highlight the potential of microarray as a technique of surgical importance. [source]

Microarray-based gene expression analysis as a process characterization tool to establish comparability of complex biological products: Scale-up of a whole-cell immunotherapy product

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009
Min Wang
Abstract Whole-cell immunotherapies and other cellular therapies have shown promising results in clinical trials. Due to the complex nature of the whole cell product and of the sometimes limited correlation of clinical potency with the proposed mechanism of action, these cellular immunotherapy products are generally not considered well characterized. Therefore, one major challenge in the product development of whole cell therapies is the ability to demonstrate comparability of product after changes in the manufacturing process. Such changes are nearly inevitable with increase in manufacturing experience leading to improved and robust processes that may have higher commercial feasibility. In order to comprehensively assess the impact of the process changes on the final product, and thus establish comparability, a matrix of characterization assays (in addition to lot release assays) assessing the various aspects of the cellular product are required. In this study, we assessed the capability of DNA-microarray-based, gene-expression analysis as a characterization tool using GVAX cancer immunotherapy cells manufactured by Cell Genesys, Inc. The GVAX immunotherapy product consists two prostate cancer cell lines (CG1940 and CG8711) engineered to secrete human GM-CSF. To demonstrate the capability of the assay, we assessed the transcriptional changes in the product when produced in the presence or absence of fetal bovine serum, and under normal and hypoxic conditions, both changes intended to stress the cell lines. We then assessed the impact of an approximately 10-fold process scale-up on the final product at the transcriptional level. These data were used to develop comparisons and statistical analyses suitable for characterizing culture reproducibility and cellular product similarity. Use of gene-expression data for process characterization proved to be a reproducible and sensitive method for detecting differences due to small or large changes in culture conditions as might be encountered in process scale-up or unanticipated bioprocess failures. Gene expression analysis demonstrated that cell products of representative lots under the same production process and at the same production scale were statistically identical. Large process changes that resulted from the artificial stress conditions used (absence of FBS and induction of hypoxia) displayed profoundly different gene expression patterns. We propose the use of simple t -test analysis in combination with the herein introduced expression ratio with mean intensity (ERMI) analysis as useful tools for process characterization by global gene expression analysis. Biotechnol. Bioeng. 2009; 104: 796,808 © 2009 Wiley Periodicals, Inc. [source]

Prediction of in vitro response to interferon-, in renal cell carcinoma cell lines

CANCER SCIENCE, Issue 4 2007
Toru Shimazui
We analyzed the correlation between interferon-, (IFN,) response and gene expression profiles to predict IFN, sensitivity and identified key molecules regulating the IFN, response in renal cell carcinoma (RCC) cell lines. To classify eight RCC cell lines of the SKRC series into three subgroups according to IFN, sensitivity, that is, sensitive, resistant and intermediate group, responses to IFN, (300,3000 IU/mL) were quantified by WST-1 assay. Microarray, followed by supervised hierarchical clustering analysis, was applied to selected genes according to IFN, sensitivity. In order to find alteration of expression profiles induced by IFN,, sequential microarray analyses were performed at 3, 6, and 12 h after IFN, treatment of RCC cell lines and mRNA expression level was confirmed using quantitative real time polymerase chain reaction. According to the sequential microarray analysis between IFN,-sensitive and -resistant line, seven genes were selected as candidates for IFN,-sensitivity-related genes in RCC cell lines. Among these seven genes, we further developed a model to predict tumor inhibition with four genes, that is, adipose differentiation-related protein, microphthalmia associated transcription factor, mitochondrial tumor suppressor 1, and troponin T1 using multiple linear regression analysis (coefficient = 0.948, P = 0.0291) and validated the model using other RCC cell lines including six primary cultured RCC cells. The expression levels of the combined selected genes may provide predictive information on the IFN, response in RCC. Furthermore, the IFN, response to RCC might be modulated by regulation of the expression level of these molecules. (Cancer Sci 2007; 98: 529,534) [source]

Rapid Screening of Lectins for Multivalency Effects with a Glycodendrimer Microarray

CHEMBIOCHEM, Issue 13 2010
Núria Parera Pera Dr.
Abstract Multivalency is an important phenomenon in protein,carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins. [source]

Fabrication of an Oriented Lectin Microarray

CHEMBIOCHEM, Issue 9 2010
Daniel C. Propheter
To tag or not to tag: An oriented lectin microarray utilizing recombinant lectins with the standard prokaryotic expression tag glutathione S -transferase has been reported. The oriented lectin microarray improves the overall sensitivity and limits of detection by site-specific orientation of recombinant lectins. This continual improvement of the lectin microarray technology enables a deeper understanding of the biological function of glycans. [source]

Developmental toxicity of estrogenic chemicals on rodents and other species

CONGENITAL ANOMALIES, Issue 2 2002
Taisen Iguchi
ABSTRACT, Antenatal sex-hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long-term changes in reproductive as well as non-reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long-term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disrupters. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT-PCR, and differential display methods, and genes related to the estrogen-independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided. [source]

Role of ancillary techniques in diagnosing and subclassifying non-Hodgkin's lymphomas on fine needle aspiration cytology

CYTOPATHOLOGY, Issue 5 2006
P. DeyArticle first published online: 8 SEP 200
Non-Hodgkin's lymphomas (NHL) are tumours of the lymphoid cells. During the process of development of lymphoid cells, neoplasia may evolve at any point. Neoplastic cells usually carry the imprint of cell of origin at the stage of origin. Various types of NHL may have similar morphology with wide variation in origin, immunophenotype and other biological features. Different ancillary laboratory techniques may help to overcome the limitations of morphology in this aspect. The commonly used ancillary techniques in lymphomas are immunocytochemistry (IC), flow cytometry, Southern blot (SB) technique, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). In addition, laser scanning cytometry (LSC) and DNA microarray technologies are in the research phase. Various laboratory techniques are used for immunophenotyping, demonstration of monoclonality, identification of chromosomal translocation, assessment of cell kinetics and expression of mRNA in the tumour cells. Flow cytometry helps in rapid immunophenotying of NHL and it has an added advantage over IC in recognizing the co-expression of CD markers. Fine needle aspiration cytology (FNAC) combined with flow immunophenotyping may help us to diagnose and subclassify certain NHLs, such as follicular lymphoma and mantle cell lymphoma, which were previously recognized as pure morphological entities. Loss of morphology is one of the important limitations of flow cytometry. LSC can overcome this limitation by studying morphology along with the immunophenotyping pattern of individual cells. Chromosomal changes in NHL can be identified by SB, PCR and FISH. Molecular diagnosis of NHL helps in diagnosis, subclassification, prognostic assessment and even in planning of therapy. DNA microarray is a relatively newer and promising technology. It gives information about the expression of several thousands of genes in a tumour in a single experiment. In the near future, FNAC combined with ancillary techniques may play a major role in diagnosis, subclassification and management of lymphomas. [source]