Home About us Contact
|
Home About us Contact | ||
|
|
||
Expression Analysis (expression + analysis)
Kinds of Expression Analysis Selected AbstractsES07 MICROARRAY GENE EXPRESSION ANALYSIS OF HUMAN ADRENOCORTICAL TUMOURSANZ JOURNAL OF SURGERY, Issue 2007P. S. H. Soon Introduction Adrenal tumours are common, occurring in 7% of patients over the age of 50. Adrenocortical carcinomas, however, are rare, with an incidence of two per million population per year. The management of adrenocortical tumours is complex, compounded by the difficulty in discriminating benign from malignant tumours using conventional histology. A molecular marker which could reliably distinguish between the two groups would be valuable in patient management. Objectives The aim of this study was to identify molecular markers which will discriminate between adrenocortical carcinomas and adenomas using microarray gene expression analysis. Methods This study used RNA from 6 normal adrenal cortices, 16 adrenocortical adenomas and 12 carcinomas. Only samples with an RNA integrity number of 7.5 or greater were used. The samples were hybridised to Affymetrix HGU133plus2.0 genechips. Data analysis was performed with Partek and affylmgui softwares. Results Using a cutoff of B > 2 and M > 2 or <,2, 217 genes were found to be significantly differentially expressed between adrenocortical adenomas and carcinomas. Of these genes, 120 were unpregulated while 97 were downregulated. Seven of these genes have been selected for validation studies with real time reverse transcription polymerase chain reaction. Conclusion In this study, we found 217 genes which were significantly differentially expressed between adrenocortical adenomas and carcinomas. With validation and further studies, these genes will provide further insight into the pathogenesis of adrenocortical tumours as well as possibly proving to be reliable discriminators between adrenocortical adenomas and carcinomas. [source] Sporulation and Germination Gene Expression Analysis of Bacillus anthracis Sterne Spores in Skim Milk under Heat and Different Intervention TechniquesJOURNAL OF FOOD SCIENCE, Issue 3 2009Y. Liu ABSTRACT:, To investigate how B. anthracis Sterne spores survive in milk under heat (80 °C, 10 min), pasteurization (72 °C, 15 s), microfiltration, and pasteurization and microfiltration, the expression levels of genes related to sporulation and germination were tested using real-time PCR assays. Twenty-seven sporulation- and germination-related genes were selected for the target genes. Our results demonstrated that gene expression levels were altered by heat and microfiltration whereas the pasteurization and pasteurization and microfiltration resulted in less alteration of gene expression. Heat activated and inhibited both sporulation- and germination-related genes, suggesting that bacterial spores underwent different molecular mechanism for heat treatments. Our results may provide some insight into the molecular mechanisms of spore survival in response to heat treatment and different intervention strategies used to treat fluid skim milk. [source] Isolation and Expression Analysis of Two Cold-Inducible Genes Encoding Putative CBF Transcription Factors from Chinese Cabbage (Brassica pekinensis Rupr.)JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 7 2006Yong Zhang Abstract Two homologous genes of the Arabidopsis C-repeat/dehydration-responsive element binding factors (CBF/DREB1) transcriptional activator were isolated by RT-PCR from Chinese cabbage (Brassica pekinensis Rupr. cv. Qinbai 5) and were designated as BcCBF1 and BcCBF2. Each encodes a putative CBF/DREB1 protein with an AP2 (Apetal2) DNA-binding domain, a putative nuclear localization signal, and a possible acidic activation domain. Deduced amino acid sequences show that BcCBF1 is very similar to the Arabidopsis CBF1, whereas BcCBF2 is different in that it contains two extra regions of 24 and 20 amino acids in the acidic domain. The mRNA accumulation profiles indicated that the expression of BcCBF1 and BcCBF2 is strongly induced by cold treatment, but does not respond similarly to dehydration or abscisic acid (ABA) treatment. However, the cold-induced accumulation of BcCBF2 mRNA was rapid but short-lived compared with that of BcCBF1. The mRNA levels of both BcCBF1 and BcCBF2 were higher in leaves than in roots when plants were exposed to cold, whereas, salt stress caused higher accumulation of BcCBF2 mRNA in roots than in leaves, suggesting that the organ specificity of the gene expression of the BcCBFs is probably stress dependent. In addition, the accumulation of BcCBF1 and BcCBF2 mRNAs was greatly enhanced by light compared with darkness when seedlings were exposed to cold. It is concluded that the two BcCBF proteins may be involved in the process of plant response to cold stress through an ABA-independent pathway and that there is also a cross-talk between the light signaling conduction pathway and the cold response pathway in B. pekinensis as in Arabidopsis. (Managing editor: Li-Hui Zhao) [source] Gene Expression Analysis in Cucumber Leaves Primed by Root Colonization with Pseudomonas chlororaphis O6 upon Challenge-Inoculation with Corynespora cassiicolaPLANT BIOLOGY, Issue 2 2004M. S. Kim Abstract: Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C. cassiicola, using plants colonized by O6. To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization. Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor. Expression of these genes was not induced by O6 colonization alone. Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection. Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation. [source] Identification and Expression Analysis of a Novel CW-Type Zinc Finger Protein MORC2 in Cancer CellsTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 6 2010Gui-Ling Wang Abstract Microrchidia2 (MORC2) is a member of the MORC protein family that is localized to both the nucleus and cytoplasm when transiently expressed in gastric cancer cells. We identified and analyzed the functional domains of MORC2, which has specific unique structural characteristics compared to the other MORC proteins. Our data showed that nuclear localization signals (NLS) of MORC2 was mainly dependent on the NLS amino acids (aa) 657,781 and cytoplasmic localization of MORC2 was attributed to the nuclear export signal (NES) aa 481,657. Moreover, the NLS appears to predominate over the NES in the localization of full-length human MORC2 indicating that MORC2 is localized mainly in the nucleus. Our results also demonstrated that the NLS (aa 657,781) and proline-rich domain within MORC2 C-terminus were required for the transcriptional repressive role in cancer cells. Anat Rec, 2010. © 2010 Wiley-Liss, Inc. [source] Application of Laser-Assisted Microdissection for Gene Expression Analysis of Mammalian Germ CellsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2010R. Kenngott With 1 figure and 2 tables Summary Laser-assisted microdissection (LAM) is an important method to provide new significant insights into many embryological processes. To understand these processes, it is important to obtain specific populations of cells from complex tissue in an efficient and precise manner and to combine with many different molecular biological methods. During the last few years, the sophistication of the techniques of LAM has increased significantly and made the procedure easy to use. New micro-extraction protocols for DNA, RNA and proteins now allow broad downstream applications in the fields of genomics, transcriptomics and proteomics. In this review, we give a short overview of the application of LAM in combination with quantitative qPCR for the analysis of gene expression in mammalian germ cells. [source] Evidence by Expression Analysis of Candidate Genes for Congenital Heart Defects in the NF1 Microdeletion IntervalANNALS OF HUMAN GENETICS, Issue 5 2005M. Venturin Summary It was recently reported that congenital heart disease is significantly more frequent in patients with NF1 microdeletion syndrome than in those with classical NF1. The outcome of congenital heart disease in this subset of patients is likely caused by the haploinsufficiency of gene/s in the deletion interval. Following in silico analysis of the deleted region, we found two genes known to be expressed in adult heart, the Joined to JAZF1 (SUZ12) and the Centaurin-alpha 2 (CENTA2) genes, and seven other genes with poorly defined patterns of expression and function. With the aim of defining their expression profiles in human fetal tissues (15th,21st weeks of gestation), expression analysis by RT-PCR and Northern blotting was performed. C17orf40, SUZ12 and CENTA2 were found to be mainly expressed in fetal heart, and following RT-PCR on mouse embryos and embryonic heart and brain at different stages of development, we found that the orthologous genes C17orf40, Suz12 and Centa2 are also expressed in early stages of development, before and during the formation of the four heart chambers. The presence of binding sites for Nkx2-5, a transcription factor expressed early in heart development, in all three mouse orthologous genes was predicted by bioinformatics, thus reinforcing the hypothesis that these genes might be involved in heart development and may be plausible candidates for congenital heart disease. [source] Targeted Gene Expression Analysis in Hemimegalencephaly: Activation of ,-Catenin SignalingBRAIN PATHOLOGY, Issue 3 2005Jia Yu MD Hemimegalencephaly (HMEG) is a developmental brain malformation characterized by unilateral hemispheric enlargement, cytoarchitectural abnormalities, and an association with epilepsy. To define the developmental pathogenesis of HMEG, the expression of 200 cell signaling, growth, angiogenic, and transcription factor genes was assayed in HMEG samples (n = 8) with targeted cDNA arrays. Differential expression of 31 mRNAs across the 4 gene families was identified in HMEG compared with control cortex. Increases in growth and transcription factor genes included JNK-1, cyclic AMP response element binding protein (CREB), and tuberin mRNAs and decreases included insulin-like growth factor-1 (IGF-1), transforming growth factor ,-3 (TGF-,3), and NFkB mRNAs. Increased expression of cyclin D1, c-myc, and WISP-1 mRNAs in HMEG suggested activation of the Wnt-1/,-catenin cascade. Western analysis demonstrated increased levels of non-phosphorylated ,-catenin, which transcriptionally activates cyclin D1 and c-myc genes, but reduced levels of Ser33/Ser37/Thr41 phospho-,-catenin, which is essential for ,-catenin-inactivation, in HMEG. Altered expression of 31 mRNAs from 4 gene families in human HMEG may lead to aberrant cell growth and hemispheric enlargement during brain development. Enhanced cyclin D1 and c-myc transcription likely reflects increased transcriptionally active ,-catenin due to decreased Ser33/Ser37/Thr41 phospho-,-catenin and suggests activation of the Wnt-1/,-catenin cascade in HMEG. [source] Molecular characterization of the Aedes aegypti odorant receptor gene familyINSECT MOLECULAR BIOLOGY, Issue 5 2007J. Bohbot Abstract The olfactory-driven blood-feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes. [source] Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2008Alberto Ferlin Abstract Introduction: Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein,coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency. Materials and Methods: Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27,41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Real-time cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2- deficient mice were studied by static and dynamic histomorphometry and ,CT, respectively. Results: Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose- and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor,ligand complex. Consistent with the human phenotype, bone histomorphometric and ,CT analyses of Rxfp2,/, mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates. Conclusions: This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis. [source] Evidences of a role for eukaryotic translation initiation factor 5A (eIF5A) in mouse embryogenesis and cell differentiationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Lucas T. Parreiras-e-Silva Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500,505, 2010. © 2010 Wiley-Liss, Inc. [source] An ortholog of MIXTA-like2 controls epidermal cell shape in flowers of ThalictrumNEW PHYTOLOGIST, Issue 3 2009Verónica S. Di Stilio Summary ,,Here, we investigated the genetic underpinnings of pollination-related floral phenotypes in Thalictrum, a ranunculid with apetalous flowers. The variable presence of petaloid features in other floral organs correlates with distinct adaptations to insect vs. wind pollination. Conical cells are present in sepals or stamens of insect-pollinated species, and in stigmas. We characterized a Thalictrum ortholog of the Antirrhinum majus transcription factor MIXTA-like2, responsible for conical cells, from three species with distinct floral morphologies, representing two pollination syndromes. ,,Genes were cloned by PCR and analysed phylogenetically. Expression analyses were conducted by quantitative PCR and in situ hybridization, followed by functional studies in transgenic tobacco. ,,The cloned genes encode R2R3 MYB proteins closely related to Antirrhinum AmMYBML2 and Petunia hybrida PhMYB1. Spatial expression by in situ hybridization overlaps areas of conical cells. Overexpression in tobacco induces cell outgrowths in carpel epidermis and significantly increases the height of petal conical cells. ,,We have described the first orthologs of AmMIXTA-like2 outside the core eudicots, likely ancestral to the MIXTA/MIXTA-like1 duplication. The conserved role in epidermal cell elongation results in conical cells, micromorphological markers for petaloidy. This adaptation to attract insect pollinators was apparently lost after the evolution of wind pollination in Thalictrum. [source] Identification and molecular analysis of candidate genes homologous to HcrVf genes for scab resistance in applePLANT BREEDING, Issue 1 2009A. Boudichevskaia Abstract The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs (called as HcrVf genes) encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on published sequences for HcrVf1 and HcrVf2 PCR primers were designed from the domain B and the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85,100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars but also from other apple scab resistance sources, such as ,Russian Seedling' R12740-7A (Vr resistance) or ,Antonovka polutorafuntovaya' (VA resistance). A series of 13 HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20,30. A considerable number of amino acid exchanges within the solvent-exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selected Vf- homologous candidate gene (called Vf2ARD) identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf2ARD is expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling suggest that this gene is scab-inducible in some resistant cultivars. Vf2ARD has been mapped on linkage group LG 1. It is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf - like locus on apple linkage group LG 1. [source] Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromesPLANT CELL & ENVIRONMENT, Issue 10 2010MARC HEIJDE ABSTRACT Cryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In this paper, we report the identification of five genes of the Cry/photolyase family (CPF) in two green algae of the Ostreococcus genus. Phylogenetic analyses were used to confidently assign three of these sequences to cyclobutane pyrimidine dimer (CPD) photolyases, one of them to a DASH-type Cry, and a third CPF gene has high homology with the recently described diatom CPF1 that displays a bifunctional activity. Both purified OtCPF1 and OtCPF2 proteins show non-covalent binding to flavin adenine dinucleotide (FAD), and additionally to 5,10-methenyl-tetrahydrofolate (MTHF) for OtCPF2. Expression analyses revealed that all five CPF members of Ostreococcus tauri are regulated by light. Furthermore, we show that OtCPF1 and OtCPF2 display photolyase activity and that OtCPF1 is able to interact with the CLOCK:BMAL heterodimer, transcription factors regulating circadian clock function in other organisms. Finally, we provide evidence for the involvement of OtCPF1 in the maintenance of the Ostreococcus circadian clock. This work improves our understanding of the evolutionary transition between photolyases and Crys. [source] White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C4 -type pyruvate orthophosphate dikinase gene (OsPPDKB)THE PLANT JOURNAL, Issue 6 2005Hong-Gyu Kang Summary We have isolated a floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion. Scanning electron microscopic analysis revealed that this abnormal endosperm consisted of loosely packed starch granules. The mutant phenotype was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophosphate dikinase (PPDK). Plants containing flo4-1 produced no OsPPDKB transcript or the OsPPDKB protein in their developing kernels and leaves. We obtained two additional alleles, flo4-2 and flo4-3, that also showed the same white-core endosperm phenotype. The flo4 kernels weighed about 6% less than wild-type ones. Starch contents in both kernel types were similar, but the total protein content was slightly higher in the mutant kernels. Moreover, lipid contents were significantly increased in the flo4 kernels. Expression analyses demonstrated that the cytosolic mRNA of OsPPDKB was induced in the reproductive organs after pollination, and greatly increased until about 10 days after fertilization. This mRNA was localized mainly in the endosperm, aleurone, and scutellum of the developing kernel. Our results suggest that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling. [source] Expression analyses and transcriptional regulation of mouse nucleolar spindle-associated protein gene in erythroid cells: essential role of NF-YBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2006Tohru Fujiwara Summary Nucleolar spindle-associated protein (NuSAP), a recently characterised microtubule-associated protein, appears to participate in cell cycle regulation. It has been demonstrated that NuSAP is expressed preferentially in the erythroid lineage in haematopoietic cells. To characterise its role in erythropoiesis, we examined the expression profile of the NuSAP gene. In fractionated murine erythroblasts, NuSAP mRNA was remarkably more abundant in the subset corresponding to immature erythroblasts (TER119+CD71high) than mature erythroblasts (TER119+CD71low), and it was significantly increased in TER119+ cells from in vivo phlebotomised mice compared with control mice. Furthermore, during erythroid maturation of mouse erythroleukaemia (MEL) cells by dimethylsulfoxide, NuSAP mRNA was increased at 24,72 h. These results suggested that the NuSAP gene might contribute to the expansion of immature erythroblast pool. The regulatory mechanism of NuSAP gene was investigated using MEL cells. Sequence analysis revealed that NuSAP promoter has four CCAAT boxes, an Sp1 element, a GATA-like element, a CACCC element, a Myb element and lacks a TATA box. Promoter analyses demonstrated that duplicated CCAAT boxes located at ,81/,85 and ,30/,34 were essential for promoter activity. Furthermore, the promoter was trans -activated by NF-YA through these elements. These results suggest that NuSAP might play an important role in erythroid proliferation under the control of NF-Y. [source] Transcriptional control of Rohon-Beard sensory neuron development at the neural plate borderDEVELOPMENTAL DYNAMICS, Issue 4 2009Christy Cortez Rossi Abstract Rohon-Beard (RB) mechanosensory neurons are among the first sensory neurons to develop, and the process by which they adopt their fate is not completely understood. RBs form at the neural plate border (NPB), the junction between neural and epidermal ectoderm, and require the transcription factor prdm1a. Here, we show that prior to RB differentiation, prdm1a overlaps extensively with the epidermal marker dlx3b but shows little overlap with the neuroectodermal markers sox3 and sox19a. Birthdating analysis reveals that the majority of RBs are born during gastrulation in zebrafish, suggesting that it is during this period that RBs become specified. Expression analysis in prdm1a and neurogenin1 mutant and dlx3b/dlx4b morpholino-injected embryos suggests that prdm1a is upstream of dlx3b, dlx4b, and neurogenin1 at the NPB. mRNA for neurogenin1 or dlx3b/dlx4b can rescue the lack of RBs in prdm1a mutants. Based on these data, we suggest a preliminary gene regulatory network for RB development. Developmental Dynamics 238:931,943, 2009. © 2009 Wiley-Liss, Inc. [source] Wnt11r is required for cranial neural crest migrationDEVELOPMENTAL DYNAMICS, Issue 11 2008Helen K. Matthews Abstract wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co-injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non,cell-autonomous manner to control neural crest migration. Developmental Dynamics 237:3404,3409, 2008. © 2008 Wiley-Liss, Inc. [source] Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterningDEVELOPMENTAL DYNAMICS, Issue 3 2004Susan C. Chapman Abstract Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway. Developmental Dynamics 229:668,676, 2004. © 2004 Wiley-Liss, Inc. [source] Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativusFEBS JOURNAL, Issue 21 2008Akihiko Tsuji Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE,cellulose, hydroxyapatite and organomercurial,Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t -butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o -aminobenzoyl-Phe-Arg-Phe(4-NO2). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination. [source] TGF-, signaling potentiates differentiation of embryonic stem cells to Pdx-1 expressing endodermal cellsGENES TO CELLS, Issue 6 2005Nobuaki Shiraki Embryonic stem (ES) cells have the capacity to differentiate to every cell type that constitutes fetal or adult tissues. To trace and quantitatively assess the differentiation of ES cells into gut endodermal cells, we used an ES cell line with the lacZ gene inserted into the pdx-1 locus. Targeted mutations of pdx-1 in mice demonstrate that pdx-1 is required for pancreatic and rostral duodenal development; therefore, pdx-1 serves as an excellent early gut regional specific marker. When these ES cells were differentiated by removal of leukemia inhibitory factor (LIF), only fractional cells turned into lacZ positive, which indicates pancreatic-duodenal differentiation. Co-cultivation of ES cells with pancreatic rudiments induced a significant increase in the proportion of lacZ positive cell numbers and this increase was further enhanced by forced expression of a chick putative endoderm inducer gene, cmix. Transforming growth factor (TGF)-,2 mimicked the effects of pancreatic rudiments and this effect was enhanced by cmix expression. Expression analysis showed over-expression of cmix induced endodermal marker genes. These data indicate that one can make use of this knowledge on molecular events of embryonic development to drive ES cells to differentiate into pdx-1 expressing endodermal cells in vitro. [source] A haplotype of the catalase gene confers an increased risk of essential hypertension in Chinese Han,HUMAN MUTATION, Issue 3 2010Zhimin Wang Abstract Our previous study in an isolated population showed an association between a genetic variant in the catalase gene (CAT) and essential hypertension (EH). This study indicates that three variants in the promoter and 5,-UTR region of CAT are predominant in Chinese Han, and they form two major haplotypes. A case,control study showed that the CATH2 haplotype confers susceptibility to EH (Pgenotype=0.0017, and Pallilc=0.00078). Subjects bearing CATH1/CATH2 and CATH2/CATH2 genotypes demonstrated a higher susceptibility to EH than CATH1/CATH1 homozygotes, with odds ratios of 1.474 and 1.625, respectively. Also, CATH1/CATH1 individuals had a later-onset age (P=0.015). Expression analysis using luciferase reporter vectors indicated that the CATH1 haplotype showed a lower transcriptional activity than the haplotype CATH2 (P<0.05 in all four cell lines), and we observed similar results in the endogenous allelic expression ratios of CATH1/CATH2 in cell lines. In contrast, most CATH1 haplotypes showed a higher transcription level than CATH2 haplotypes (10 out of 11 or 90.9%) in blood from normal individuals (P<0.01). We therefore hypothesize that CATH1 and CATH2 may play alternating roles at different level of oxidative stress. Hum Mutat 31:272,278, 2010. © 2009 Wiley-Liss, Inc. [source] Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiaeINSECT MOLECULAR BIOLOGY, Issue 6 2003P. X. Xu Abstract We performed a genome-wide analysis for candidate odorant-binding protein (OBP) genes in the malaria vector Anopheles gambiae (Ag). We identified fifty-seven putative genes including sixteen genes predicted to encode distinct, higher molecular weight proteins that lack orthologues in Drosophila. Expression analysis indicates that several of these atypical AgOBPs are transcribed in chemosensory organs in adult and immature stages. Phylogenetic analysis of the Anopheles and Drosophila OBP families reveals these proteins fall into several clusters based on sequence similarity and suggests the atypical AgOBP genes arose in the mosquito lineage after the divergence of mosquitoes and flies. The identification of these AgOBP genes is the first step towards determining their biological roles in this economically and medically important insect. [source] Alteration of subcellular and cellular expression patterns of cyclin B1 in renal cell carcinoma is significantly related to clinical progression and survival of patientsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Stephen O. Ikuerowo Abstract Cyclin B1, identified as a regulator of late cell cycle, is involved in the development and progression of a variety of human malignancies. To clarify the role of cyclin B1 in the pathogenesis and prognosis of renal cell carcinoma (RCC), protein expression was compared with clinicopathological characteristics of patients as well as the long-term survival after surgical therapy. Expression analysis was carried out by immunohistochemistry and tissue microarray analysis. The microarrays that represented the primary tumors, their invasion front and normal peritumoral renal parenchyma contained 753 tissue cores obtained from 251 randomly selected nephrectomy specimens. Immunopositivity within the primary tumors was significantly associated with tumor stage (pT) (p < 0.01), lymph node status (pN) (p < 0.01) as well as the presence of systemic metastatic disease (p = 0.01). Subcellular expression in the cytoplasm of tumor cells significantly correlated with pT (p = 0.02) and pN (p = 0.03). When peritumoral tissue samples exhibited a relative amount of <10% of positively reacting epithelial cells, cyclin B positivity was identified to predict long-term survival of patients in univariate analysis (p < 0.01) whereas borderline significance was observed in multivariate statistical analysis (p = 0.05). Increased intratumoral cyclin B1 positivity and aberrant localization of signals within the cytoplasm of tumor cells is positively correlated with the tendency towards tumor progression, indicating the significant role of cyclin B1 in the development and pathogenesis of RCC. The result of uni- and multivariate statistical analysis suggests the prognostic value of cyclin B1 for RCC patients. © 2006 Wiley-Liss, Inc. [source] Diversification in MHC class II invariant chain-like proteins among fishesJOURNAL OF APPLIED ICHTHYOLOGY, Issue 4 2004M. Sakai Summary The major histocompatibility complex (MHC) class II invariant chains are important for an efficient and complete presentation of antigens by MHC class II molecules. Invariant chain-like proteins (Iclp) 1 and 2 were identified by expressed sequence tag analysis from cDNA library of common carp head kidney (HK) stimulated with concanavalin A and lipopolysaccharide. The sequences were 1043 and 1016 bp in length encoding 234 and 198 amino acid proteins, respectively. Based on their predicted structure, the genes harboured transmembrane domain (TMD) and Tg (thyroglobulin) type 1 domains. Expression analysis revealed that both genes were expressed in normal tissues of HK, intestine, brain and gill. By database search, similar homologues were found in Atlantic salmon, fugu and catfish. Phylogenetic and alignment analysis indicate diversity among fish Iclps. [source] Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2008Alberto Ferlin Abstract Introduction: Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein,coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency. Materials and Methods: Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27,41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Real-time cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2- deficient mice were studied by static and dynamic histomorphometry and ,CT, respectively. Results: Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose- and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor,ligand complex. Consistent with the human phenotype, bone histomorphometric and ,CT analyses of Rxfp2,/, mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates. Conclusions: This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis. [source] The OsDHODH1 Gene is Involved in Salt and Drought Tolerance in RiceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2009Wen-Ying Liu Abstract In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid (ABA), but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coli cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODH1 -antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought. [source] Genetic Analysis and Molecular Mapping of a Rolling Leaf Mutation Gene in RiceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 12 2007Ji-Cai Yi Abstract A rice mutant with rolling leaf, namely ,- rl, was obtained from M2 progenies of a native indica rice stable strain Qinghuazhan (QHZ) from mutagenesis of dry seeds by ,-rays. Genetic analysis using the F2 population from a cross between this mutant and QHZ indicated the mutation was controlled by a single recessive gene. In order to map the locus for this mutation, another F2 population with 601 rolling leaf plants was constructed from a cross between ,- rl and a japonica cultivar 02428. After primary mapping with SSR (simple sequence repeats) markers, the mutated locus was located at the short arm of chromosome 3, flanked by RM6829 and RM3126. A number of SSR, InDel (insertion/deletion) and SNP (single nucleotide polymorphism) markers within this region were further developed for fine mapping. Finally, two markers, SNP121679 and InDel422395, were identified to be flanked to this locus with genetic distances of 0.08 cM and 0.17 cM respectively, and two SNP markers, SNP75346 and SNP110263, were found to be co-segregated with this locus. These results suggested that this locus was distinguished from all loci for the rolling leaf mutation in rice reported so far, and thus renamed rl10(t). By searching the rice genome database with closely linked markers using BLAST programs, an e -physical map covering rl10(t) locus spanning about a 50 kb region was constructed. Expression analysis of the genes predicted in this region showed that a gene encoding putative flavin-containing monooxygenase (FMO) was silenced in ,- rl, thus this is the most likely candidate responsible for the rolling leaf mutation. [source] Expression analysis of genes induced in barley after chemical activation reveals distinct disease resistance pathwaysMOLECULAR PLANT PATHOLOGY, Issue 5 2000Katrin Beßer Salicylic acid (SA) and its synthetic mimics 2,6-dichloroisonicotinic acid (DCINA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), protect barley systemically against powdery mildew (Blumeria graminis f.sp. hordei, Bgh) infection by strengthening plant defence mechanisms that result in effective papillae and host cell death. Here, we describe the differential expression of a number of newly identified barley chemically induced (BCI) genes encoding a lipoxygenase (BCI-1), a thionin (BCI-2), an acid phosphatase (BCI-3), a Ca2+ -binding EF-hand protein (BCI-4), a serine proteinase inhibitor (BCI-7), a fatty acid desaturase (BCI-8) and several further proteins with as yet unknown function. Compared with SA, the chemicals DCINA and BTH were more potent inducers of both gene expression and resistance. Homologues of four BCI genes were detected in wheat and were also differentially regulated upon chemical activation of disease resistance. Except for BCI-4 and BCI-5 (unknown function), the genes were also induced by exogenous application of jasmonates, whereas treatments that raise endogenous jasmonates as well as wounding were less effective. The fact that BCI genes were not expressed during incompatible barley,Bgh interactions governed by gene-for-gene relationships suggests the presence of separate pathways leading to powdery mildew resistance. [source] Mice deficient for RNA-binding protein brunol1 show reduction of spermatogenesis but are fertileMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 11 2007Arvind Dev Abstract RNA-binding proteins are involved in post-transcriptional processes like mRNA stabilization, alternative splicing, and transport. Brunol1 is a novel mouse gene related to elav/Bruno family of genes encoding for RNA-binding proteins. We report here the expression and functional analysis of murine Brunol1. Expression analysis of Brunol1 during embryogenesis by RT-PCR showed that Brunol1 expression starts at 9.5 dpc and continues to the later stages of embryonic development. In adult mice, the Brunol1 expression is restricted to brain and testis. We also analyzed the Brunol1 expression in testes of different mutants with spermatogenesis defects: W/WV, Tfm/y, Leyl,/,, olt/olt, and qk/qk. Brunol1 transcript was detectable in Leyl,/,, olt/olt, and qk/qk mutant but not in W/WV and Tfm/y mutants. We also showed by transfection of a fusion protein of green fluorescent protein and Brunol1 protein into NIH3T3 cells, that Brunol1 is localized in cytoplasm and nucleus. In order to elucidate the function of the Brunol1 protein in spermatogenesis, we disrupted the Brunol1 locus in mouse by homologous recombination, which resulted in a complete loss of the Brunol1 transcript. Male and female Brunol1+/, and Brunol1,/, mice from genetic backgrounds C57BL/6J,×,129/Sv hybrid and 129X1/SvJ when inbred exhibited normal phenotype and are fertile, although the number and motility of sperms are significantly reduced. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. Collectively our results demonstrate that Brunol1 might be nonessential protein for mouse embryonic development and spermatogenesis. Mol. Reprod. Dev. 74: 1456,1464, 2007. © 2007 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||