Cloning and expression pattern analysis of chalcone synthase gene in Microcos paniculata / 中草药

Objective To clone the full-length cDNA of MpCHS and analyze its expression pattern in different parts of Microcos paniculata and different growth periods of M. paniculata leaves. Methods Based on the transcriptome data of M. paniculata, we designed specific primers for MpCHS gene. The full-length cDNA of MpCHS was amplified by PCR and the positive clones were then sequenced, analyzed, and constructed prokaryotic expression vector. The bioinformatics analysis of MpCHS was also performed. Meanwhile, the mRNA expression of MpCHS was detected using real-time quantitative PCR. Results The relative molecular mass was 42 700, and its theoretical isoelectric point was 6.11, with three conserved functional active sites (165 C, 304 H, and 337 N) of the CHS family proteins and the tag sequence of RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Phylogenetic tree analysis showed that MpCHS had close relationship with woody plants such as cocoa and upland cotton. We successfully cloned the full-length cDNA of MpCHS (GenBank: KY472608). It had an ORF of 1 176 bp which encoded a protein of 391 amino acid residues. RT-qPCR results showed that MpCHS was expressed in all parts of M. paniculata and its expression in leaves was gradually decreased along with its development. Conclusion MpCHS is cloned from M. paniculate for the first time, and the gene expression pattern of MpCHS in different parts of M. paniculata and different growth periods of M. paniculata leaves was analyzed. This study facilitates the further purification and functional validation of MpCHS protein and provides reference for further analysis of flavonoids biosynthesis pathway in M. paniculata.

Significantly reduced function of T cells in patients with acute arterial thrombosis / 老年心脏病学杂志（英文版）

Objectives To explore the intrinsic factors related to the pathogenesis of acute arterial thrombosis (AAT) and to elucidate the patho-genesis of AAT on the basis of differentially expressed genes. Methods Patients with acute myocardial infarction (AMI), stable angina (SA) and healthy controls (n=20 per group) were recruited, and the whole human genome microarray analysis was performed to detect the dif-ferentially expressed genes among these subjects. Results Patients with AMI had disease-specific gene expression pattern. Biological func-tional analysis showed the function of T cells was significantly reduced, the mitochondrial metabolism significantly decreased, the ion me-tabolism was abnormal, the cell apoptosis and inflammatory reaction increased, the phagocytosis elevated, the neutrophil-mediated immunity increased and the post-traumatic repair of cells and tissues increased in AMI patients. The biological function in SA group and healthy con-trols remained stable and was comparable. Conclusions The reduced function of T cell gene models in AAT showed the dysfunction of the immune system. The pathogenesis of AAT may be related to the inflammatory reaction after arterial intima infection caused by potential pathogenic microorganisms.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-gamma in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

A novel lipocalin homologue from the venom gland of Deinagkistrodon acutus similar to mammalian lipocalins

Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo), isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5 percent. Primary structures of d-lipo carried three structurally conserved regions (SCR) showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS) in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP) cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.(AU)

cDNA microarray in the gene expression pattern in lymphatic metastasis of colon carcinomas / 肿瘤研究与临床

Objective To analyze differential gene expression profiles by cDNA microarray in colon carcinomas with or without lymphatic metastasis. Methods cDNA microarray was prepared by spotting polymerase chain reaction (PCR) products of 16 000 human genes onto specially treated glass slides. The cDNA probes were prepared by labeling cancer tissue mRNA and lymphatic metastasis tissue mRNA with Cy3-dUTP and Cy5- dUTP through reverse transcription. The mixed probes were ,then hybridized to the cDNA microarray. The chips were scanned by Agilent fluorescence scanner and analyzed by gene Pix QuantArray. Results Among the 16 000 target genes, 999 genes were screened out for differences in gene expression level in the cases with colon carcinoma and lymphatic metastasis, among which 537 were up-regulated and 462 down-regulated. There were many genes evolved in the metastasis of colon carcinoma, including oncogenes, tumor-suppressor genes, adhesion molecular, matrix metalloproteinases, signal transduction factors, metabolism, immune associated genes, etc. Conclusion The genes, being closely associated with carcinoma metastasis, could be considered as potential markers to predict metastasis and targets for antimetastasis intervention.

Molecular Characterization and Expression Analysis of Five Novel Genes Encoding Proline-rich Proteins in Cotton(Gossypium hirsutum) / 生物化学与生物物理进展

Proline-rich cell wall proteins are widely spread in plants and are believed to function by modeling the architecture of the cell wall surrounding specific cell types. Five genes encoding proline-rich proteins were isolated from cotton cDNA libraries. The most common characteristic of these proteins is the abundant proline residues that occur in repeating motifs of at least two consecutive Pros. Based on amino acid composition, repetitive motifs and domain organization, the five members can be divided into two subgroups: one group similar to common PRPs including GhPRP3, GhPRP6, GhPRP5 and GhPRP4 was composed of two domains, an N-terminal hydrophobic domain (or signal peptide) followed by a proline-rich domain containing different proline-rich repetitive motifs; the other group different from common PRPs including GhPRPL lies in it contains an N-terminal hydrophilic domain, eight repetitive copies of pentapeptide (similar to PPKKE) lies in the C-terminal domain. Expression studies of the six GhPRPs have been carried out by quantitative realtime RT-PCR. The results showed that GhPRP3 and GhPRP5 were preferentially expressed in 10 dpa fiber, little transcripts was detected in other tissues examined. GhPRPL highly expressed in cotyledons, whereas smaller or negligible amounts of its transcripts were detected in other tissues. The remaining two genes, GhPRP4 and GhPRP6, were expressed in all the tissues analysed, but their transcript level is different. GhPRP4 mRNA is most abundant in hypocotyls, and then in anther, while GhPRP6 expressed highly in fiber, and then in 10 dpa ovule. Furthermore, the results showed that the fiber-specific GhPRP3 and GhPRP5 were also developmentally regulated, suggesting that the genes may play important roles during cotton fiber development.

Study of Apoptosis and Gene Expression Pattern Related to Human Degenerative Intervertebral Disc Tissue / 中国康复理论与实践

@#ObjectiveTo investigate the relationship between the degenerative mechanisms of lumbar intervertebral disc (LID) and apoptosis.MethodsThe total RNAs were isolated from human LID tissues. Both the mRNAs from the degeneration and normal LID were reversely transcribed to the cDNAs. The cDNAs were labeled with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed by computer image analysis. The apoptotic status and the expression of Bcl-2 and Bax in 12 cases of degenerative LID and 10 cases of normal LID were detected with TdT-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemistry methods.ResultsAmong the 4096 targets, there were 10 genes related to apoptosis. The expression related to Bax protein gene was up-regulated and it was down-regulated for Bcl-2 protein. In group of normal LID, the average apoptotic index (AI) was (24.897±3.620); percentage of Bcl-2 positive cells was (31.440±4.150)%; percentage of Bax positive cells was (29.372±2.588)%, average optical density (OD) values of positive particles were (0.183± 0.010 ), ( 0.203 ±0.012) and (0.169±0.005) respectively. In group of degenerative LID, the average AI was (49.232±3.440); percentage of Bcl-2 positive cells was (18.239±2.470)%; percentage of Bax positive cells was (52.349±3.764)%; average OD values of positive particles were (0.152±0.003), (0.310±0.008) and (0.262±0.014) respectively. There were significantly differences in AI and expressions of Bcl-2 and Bax proteins between normal LID and degenerative LID (P<0.05).ConclusionCell apoptosis plays an important role in the process of LID degeneration. Both Bcl-2 and Bax take part in the occurrence and progression of LID.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human / 中国康复理论与实践

@#ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

Screening for Causative Genes Involved in Children with Minimal Change Nephritic Syndrome / 实用儿科临床杂志

Objective To screen for the causative genes involved in the occurrence and development of minimal changes nephritic syndrome(MCNS) and to furtherly assist the genetic diagnosis and treatment of MCNS.Methods Human genome U133 Array Set from Affymetrix Inc was used to evaluate gene expression patterns in peripheral blood mononuclear cells(PBMC) isolated from 7 children with primary MCNS and 7 age-matched health volunteers.Reverse transcription-polymerase chain reaction(RT-PCR) and real-time PCR were performed to identify the findings of gene chip.Results Of 33 000 genes detected,969 genes showed significant difference between children with(MCNS) and healthy volunteers;552 genes were up-regulated,while 417 genes down-regulated significantly.Findings from RT-PCR and real-time PCR were consistent with those of gene chip.Conclusions Gene chip of expression patterns is a powerful method to detect expression difference of genes correlated with MCNS.Occurrence and development of MCNS can be a complicated process that many correlative genes may participate in.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human / 中国康复理论与实践

Objective To investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. Methods The PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. Results Among the 4096 targets,there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases,comprising 298 up-regulated and 358 down-regulated ones. Conclusion DNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

Gene Expression Pattern Analysis via Latent Variable Models Coupled with Topographic Clustering

We present a latent variable model-based approach to the analysis of gene expression patterns, coupled with topographic clustering. Aspect model, a latent variable model for dyadic data, is applied to extract latent patterns underlying complex variations of gene expression levels. Then a topographic clustering is performed to find coherent groups of genes, based on the extracted latent patterns as well as individual gene expression behaviors. Applied to cell cycle regulated genes of the yeast Saccharomyces cerevisiae, the proposed method could discover biologically meaningful patterns related with characteristic expression behavior in particular cell cycle phases. In addition, the display of the variation in the composition of these latent patterns on the cluster map provided more facilitated interpretation of the resulting cluster structure. From this, we argue that latent variable models, coupled with topographic clustering, are a promising tool for explorative analysis of gene expression data.

Gene expression pattern of cervical intervertebral disc degeneration in rats / 第二军医大学学报

Objective: To investigate the gene expresssion changes in normal and degeneration cervical intervertebral disc in rats, providing information for clinical diagnosis and treatment of cervical intervertebral disease.Methods: The models of cervical intervertebral disc degeneration in rats caused by imbalance between the dynamic and static forces was established.cDNA microarray chips containing 512 cDNAs were used to investigate the gene expression pattern in cervical intervertebral disc of rat.The results were scanned and analyzed by computer image analysis,and then subjected to standardization,ratio and clustering analysis.Results: Eighteen genes expressed in cervical intervertebral disc were screened out,comprising 11 up regulated and 7 down regulated ones,respectively.This genes expression in cervical spondylosis was similar to other reports.Conclusion: Signal transduction pathways plays an important role in the process of cervical intervertebral disc degeneration.Investigation on the gene expression pattern is helpful for studying degeneration of cervical intervertebral disc pathogenesis.