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At least 217 records · Page 12Linked to original sources

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.

Animals↗

A murine dopamine neuron-specific cDNA library and microarray: increased COX1 expression during methamphetamine neurotoxicity.

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

3,4-Dihydroxyphenylacetic Acid↗

Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis.

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.

Animals↗

Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of postnatal-but not embryonic-mouse dorsal root ganglion cells in vitro, despite the fact that its receptors are expressed at both ages. To understand this difference, we have performed an oligonucleotide microarray experiment. We found that several hundred genes were regulated between embryonic and postnatal stages, and that several important classes of genes were differentially regulated by GDNF treatment, including genes related to translation and to phenotypic specification and maturation. Interestingly, a set of genes related to cell adhesion, cytoskeleton and cellular morphology were consistently down-regulated by GDNF, suggesting a previously uncharacterized role for GDNF in repressing neurite growth and/or branching. This nuclear program initiated by GDNF was functionally confirmed in cultures of embryonic wild-type neurons sustained with nerve growth factor and in bax(-/-) neurons that survive in the absence of trophic support.

Aging↗

Molecular characterization of a metastatic neuroendocrine cell cancer arising in the prostates of transgenic mice.

The features and functions of prostatic neuroendocrine (NE) cells remain ill-defined. Neuroendocrine differentiation (NED) in adenocarcinoma of the human prostate (CaP) is associated with more aggressive disease, but the underlying mediators are poorly understood. We examined these issues in transgenic mice that utilize regulatory elements from the cryptdin-2 gene (Defcr2) to express simian virus 40 large T antigen (TAg) in prostatic NE cells. CR2-TAg mice develop prostatic intraepithelial neoplasia at 8 weeks of age, 1 week after the onset of TAg expression. An invasive phase follows 2-4 weeks later, with lymph node, liver, lung, brain, and bone metastases appearing within 16 weeks. DNA microarray studies revealed 122 mRNAs that were increased >/=2-fold in duplicate assays of 16-week-old CR2-TAg versus normal prostates. Thirty two transcripts encode proteins associated with neurons and endocrine cells (e.g. basic helix loop helix, SRY-related high mobility group box and sine-oculis homeobox transcription factors, Hu RNA-binding proteins, neuronatin, Racgap1, collapsin response mediator protein-1, synaptotagmin-1, proprotein convertase, and secretogranins). Follow-up studies of candidate mediators and biomarkers of differentiation/growth in the microarray data set involved real time quantitative reverse transcriptase-PCR assays of laser capture microdissected NE cells from CR2-TAg prostates plus liver metastases, and immunohistochemical comparisons of transgenic mouse prostates and 35 human CaP samples. Our findings include (a) expression of the bHLH mouse achaete-scute homolog (mASH1) in normal and CR2-TAg NE cells and foci of NED in human CaP, (b) glutamic acid decarboxylase and its product (gamma-aminobutyric acid) in neoplastic NE cells juxtaposed next to cohorts of normal gamma-aminobutyric acid receptor expressing secretory cells (a potential route for paracrine interactions between these two epithelial lineages), and (c) aromatic l-amino-acid decarboxylase, but not its dopamine/serotonin products, in CR2-TAg NE cells and NED. These results underscore the value of CR2-TAg mice for characterizing normal NE cell biology and tumorigenesis.

Animals↗

A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links.

We describe a vertebrate hyaluronan and proteoglycan binding link protein gene family (HAPLN), consisting of four members including cartilage link protein. The encoded proteins share 45-52% overall amino acid identity. In contrast to the average sequence identity between family members, the sequence conservation between vertebrate species was very high. Human and mouse link proteins share 81-96% amino acid sequence identity. Two of the four link protein genes (HAPLN2 and HAPLN4) were restricted in expression to the brain/central nervous system, while one of the four genes (HAPLN3) was widely expressed. Genomic structures revealed that all four HAPLN genes were similar in exon-intron organization and were also similar in genomic organization to the 5' exons for the CSPG core protein genes. Strikingly, all four HAPLN genes were located immediately adjacent to the four CSPG core protein genes creating four pairs of CSPG-HAPLN genes within the mammalian genome. Furthermore, the two brain-specific HAPLN genes (HAPLN2 and HAPLN4) were physically linked to the brain-specific CSPG genes encoding brevican and neurocan, respectively. The tight physical association of the HAPLN and CSPG genes supports a hypothesis that the first HAPLN gene arose as a partial gene duplication event from an ancestral CSPG gene. There is some degree of coordinated expression of each gene pair. Collectively, the four HAPLN genes are expressed by most tissue types, reflecting the fundamental importance of the hyaluronan-dependent extracellular matrix to tissue architecture and function in vertebrate species. Comparison of the genomic structures for the HAPLN, CSPG genes and other members of the link module superfamily provide strong support for a common evolutionary origin from an ancestral gene containing one link module encoding exon.

Animals↗

Transforming growth factor-beta1-modulated cerebral gene expression.

Transforming growth factor-beta1 (TGF-beta1) plays a central role in the response of the brain to different types of injury. Increased TGF-beta1 has been found in the central nervous system of patients with acute or chronic disorders such as stroke or Alzheimer disease. To further define the molecular targets of TGF-beta1 in cerebral tissues, a selection of high-density cDNA arrays was used to characterize the mRNA expression profile of 7,000 genes in transgenic mice overexpressing TGF-beta1 from astrocytes as compared with the wild-type line. Selected findings were further evaluated by reverse transcription-polymerase chain reactions from independent transgenic and wild-type mice. Furthermore, the expression pattern of seven selected genes such as Delta-1, CRADD, PRSC-1, PAI-1, Apo-1/Fas, CTS-B, and TbetaR-II were confirmed in either cultured cortical neurons or astrocytes following TGF-beta1 treatment. The authors' observations enlarge the repertoire of known TGF-beta1-modulated genes and their possible involvement in neurodegenerative processes.

Animals↗

Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor.

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.

Apoptosis↗

Multiple cross and inbred strain haplotype mapping of complex-trait candidate genes.

Identifying complex-trait candidate genes after initial low-resolution mapping has proven to be a difficult and labor-intensive undertaking, usually requiring years to develop and analyze congenic strains. As a result, to date, few complex-trait genes have been discovered. Recently it was suggested that SNP haplotype analysis in inbred strains might be useful for mapping of complex traits. In this study, we have combined medium-resolution haplotype mapping with multiple experimental cross-mapping experiments to reduce the number of potential candidate genes in a complex-trait candidate interval. Coincident mapping of a modifier gene in multiple experimental crosses using different inbred strains is consistent with the common inheritance of a modifier allele. A haplotype map was developed in four inbred strains of mice used in our complex-trait mapping crosses across the proximal 10 cM of proximal Chromosome 19 to identify haplotype blocks that segregate appropriately. Only ~23 out of >400 genes met this criteria. This strategy coupled with tissue and expression arrays, as well as our recently described common pathway analysis to reduce the number of high-priority candidates, may provide a rapid, efficient method to identify and prioritize complex-trait candidate genes without requiring construction of congenic mouse strains.

Animals↗

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis.

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor-stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

Biomarkers, Tumor↗

Regional and total body bone mineral content, bone mineral density, and total body tissue composition in children 8-16 years of age.

Normative values for total body bone mineral content (TBBM) and total body bone mineral density (TBMD) were derived from measurements on 234 children 8-16 years of age. In addition, bone mineral content (BMC) and bone mineral density (BMD) values for selected regions of interest and soft tissue (bone free lean and fat) for the total body are presented. Bone mineral and soft tissue values were determined by dual energy X-ray absorptiometry (DXA) using a Hologic QDR-2000 in the array mode. Results of a stepwise multiple regression analysis revealed a significant correlation between bone-free lean tissue (BFLT) and BMD (r2 = 0.80) in girls. Adding age to the equation accounted for an additional 2% of the variance (P < 0.05) and height accounted for another 1% of the variance (P < 0.05). Body weight and fat tissue (FT) did not account for any additional variance. In boys BFLT correlated significantly with BMD (r2 = 0.75; P < 0.05); none of the other predictor variables accounted for additional variance. No significant differences were found in TBBM or TBMD between boys and girls at any age. There was a significant overall gender effect for only three regions of interest. Boys had greater BMC in the head region and had greater BMD in the upper limbs, but post hoc analysis revealed no significant differences for any specific age groups. Girls had greater overall BMD in the pelvis, but this difference was only significant at the 15-16-year age group. The changes in BFLT and FT over the age ranges were consistent with the growth literature.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗

Regulation of insulin-like growth factor-binding proteins in the baboon (Papio anubis) uterus during early pregnancy.

The baboon uterus begins to synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) in the deep glands of the late secretory endometrium, and this protein then becomes the major secretory product of the term decidua. We hypothesized that the placenta and/or conceptus may regulate the synthesis and secretion of IGFBP-1 by decidualized stromal cells during pregnancy. To test this hypothesis, tissue was obtained from pregnant baboons on days 18, 25, and 32 postovulation. The uterus was separated into three regions: RI (directly below the implantation site), RII (adjacent to the implantation site), and RIII (opposite the implantation site). Portions of the tissue were fixed in Bouin's solution for immunocytochemistry, and the remainder was subdivided into functionalis, basalis, and myometrium and subjected to organ explant culture. The placenta was fixed or cultured separately. Ligand blot analysis of functionalis medium showed that the major IGFBP had a mol wt (Mr) of 29,000-31,000; however, a doublet of 37,000-43,000 Mr and a band at 24,000 Mr were also present. The functionalis from all regions expressed the majority of the IGFBPs, but basalis from RI tissue also secreted the same array of IGFBPs on days 25 and 32. Ligand blot analysis of placental medium proteins revealed a doublet at Mr 37,000-43,000 on days 25 and 32, but not on day 18. Immunoprecipitation followed by ligand blot analysis of medium proteins using polyclonal antibodies to IGFBP-1 and IGFBP-2 and -3 confirmed that IGFBP-1 and -2 were the predominant products of the endometrium and decidua, while IGFBP-3 was synthesized by the placenta. Immunocytochemistry with a monoclonal antibody to IGFBP-1 demonstrated intense glandular epithelial staining in all regions on days 18, 25, and 32. Stromal staining for IGFBP-1 was first evident on day 25 and was only present in stromal cells in intimate contact with the trophoblastic tissue. By day 32, IGFBP-1 expression was not limited to the endometrial-trophoblastic junction, but extended to the deeper stromal cells and included the perivascular regions. IGFBP-1 staining was most intense in RI, but stromal cells at the luminal surface and those surrounding the spiral arteries also showed some staining in RII and RIII on day 32. These studies suggest that the baboon placenta and/or conceptus regulate IGFBP expression in the uterine endometrium during the initial stages of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Classification and expression analysis of Arabidopsis F-box-containing protein genes.

F-box proteins regulate diverse cellular processes, including cell cycle transition, transcriptional regulation and signal transduction, by playing roles in Skp1p-cullin-F-box protein (SCF) complexes or non-SCF complexes. F-box proteins are encoded by a large gene family. Our database search revealed that at least 568 F-box protein genes are present in the Arabidopsis thaliana (Arabidopsis) genome. Domain search analysis using SMART and Pfam-A databases revealed that 67 of the F-box proteins contained Kelch repeats and 29 contained leucine-rich repeats (LRRs). Interestingly only two F-box proteins contained WD40 repeats that are found in many F-box proteins of other organisms. Kelch repeats, LRRs and WD40 repeats are implicated in protein-protein interactions. This analysis also resulted in the finding of several unique functional domains; however, 448 of the F-box proteins did not contain any known domains. Therefore, these proteins were used to search the Pfam-B database to find novel domains, and three putative ones were found. These domain search analyses led us to classify the Arabidopsis F-box proteins into at least 19 groups based on their domain structures. Macro array analysis showed that several F-box protein genes are expressed in a tissue-specific manner.

Arabidopsis↗

Gene expression analysis in microdissected renal tissue. Current challenges and strategies.

The architecture and compartmentalization of the kidney has stimulated the development of an array of microtechniques to study the functional differences between the distinct nephron segments. With the vast amounts of genomic sequence data now available, the groundwork has been laid for a comprehensive characterization of the molecular pathways defining the differences in nephron function. With the development of sensitive gene expression techniques the tools for a comprehensive molecular analysis of specific renal microenvironments have been provided: Quantitative RT-PCR technologies now allow the analysis of specific mRNAs from as little as single microdissected renal cells. A more global view of gene expression regulation is a logical development from the application of large scale profiling techniques. In this review, we will discuss the power and pitfalls of these approaches, including their potential for the functional characterization of nephron heterogeneity and diagnostic application in renal disease.

Dissection↗

Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes.

The effects of transgene delivery by adenoviral vectors were studied by probing a 588 gene, mouse cDNA array with mRNA derived from infected liver. The liver tissues were obtained from naive mice and mice infected with replication-deficient adenovirus, adenovirus expressing transforming growth factor beta1 (TGFbeta1), and adenovirus expressing connective tissue growth factor (CTGF). Expression of 98 genes was detected in the array analysis. The increased expression of the transcripts for Stat1, gamma interferon-induced monokine (MIG) and interferon regulatory factor 1 (IRF1) clearly demonstrated the immune response induced by infection with a first generation, replication-incompetent adenovirus. In vivo expression of TGFbeta1 led to a down-regulation of genes involved in the immune response. The increased expression of u-PAR1, laminin receptor and BMP-1 confirms the importance of CTGF and TGFbeta1 in angiogenesis, and tissue repair. Expression of the serine protease inhibitors, Spi 2.4 and Spi 2, is also increased in response to AdTGFbeta1 and AdCTGF.

Adenoviridae↗

Gene expression profiling of cancer by use of DNA arrays: how far from the clinic?

DNA arrays allow the simultaneous analysis of expression levels for thousands of genes in normal and pathological tissues and hold great promise in molecular medicine, notably in cancer research. The great biological and clinical diversity present in human tumours is poorly characterised by the current classification systems. DNA arrays can provide a better understanding of oncogenesis, leading to improvements in cancer management. First, the identification of new target genes and pathways will allow the development of specific molecular-based anticancer drugs. Secondly, expression profiles will permit tumour classification in more homogeneous diagnostic and prognostic groups, as well as the identification of new clinically and biologically relevant tumour subclasses. Here, we review the technology and present some cancer studies with promising results. Finally, we discuss some of the issues that must be resolved in the near future, so that DNA arrays can fulfil the aims mentioned above.

Gene Expression Profiling↗

Molecular techniques for studying gene expression in carcinogenesis.

Many genes and signaling pathways controlling cell proliferation, death, differentiation, and genomic integrity are involved in cancer development. Various methods are available for detection and quantification of messenger RNA. Older methods such as Northern blots, nuclease protection, plaque hybridization, and slot blots suffer from being inherently serial, measure a single mRNA at a time, or being difficult to automate. New techniques for analysis of gene expression include: (a) comprehensive open systems such as serial analysis of gene expression (SAGE), differential display (DD) analysis, RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphism (AFLP), total gene expression analysis (TOGA), and use of internal standard competitive template primers (CTs) in a quantitative multiplex RT-PCR method [StaRT-(PCR)], and (b) focused closed systems such as: high density cDNA filter hybridization (HDFCA) analysis, suppression subtractive hybridization (SSH), differential screening (DS), several forms of high-density cDNA arrays, or oligonucleotide chips, and tissue microarrays. Sometimes, a combination of these systems is used to enhance the sensitivity and specificity of the assays. While closed systems are excellent for the initial screening of large number of sequences, the value of the information generated is generally limited to an often arbitrarily chosen known sequence. On the other hand, only the open system platform has the potential to evaluate the expression patterns of tens of thousands of genes that have not yet been cloned or partially sequenced in a quantitative manner. A cost analysis of the most commonly used expression technologies is provided. A method for purifying tumors from surrounding stroma and normal tissue employing laser microdissection, and subsequent RNA isolation/amplification from few cells employing sensitive kits are also discussed.

Cell Transformation, Neoplastic↗

Changes in tumorigenesis- and angiogenesis-related gene transcript abundance profiles in ovarian cancer detected by tailored high density cDNA arrays.

BACKGROUND: Complementary DNA array analysis of gene expression has a potential application for clinical diagnosis of disease processes. However, accessibility, affordability, reproducibility of results, and management of the data generated remain issues of concern. Use of cDNA arrays tailored for studies of specific pathways, tissues, or disease states may render a cost- and time-effective method to define potential hallmark genotype alterations. MATERIALS AND METHODS: We produced a 332-membered human cDNA array on nylon membranes tailored for studies of angiogenesis and tumorigenesis in reproductive disease. We tested the system for reproducibility using a novel statistical approach for analysis of array data and employed the arrays to investigate gene expression alterations in ovarian cancer. RESULTS: Intra-assay analysis and removal of agreement outliers was shown to be a critical step prior to interpretation of cDNA array data. The system revealed highly reproducible results, with intermembrane coefficient of reproducibility of +/- 0.98. Comparison of placental and ovarian sample data confirmed expected differences in angiogenic profiles and tissue-specific markers, such as human placental lactogen (hPL). Analysis of expression profiles of five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples revealed an overall increase in angiogenesis-related markers, including vascular endothelial growth factor (VEGF) and angiopoietin-1 in the diseased tissue. These were accompanied by increases in immune response mediators (e.g. HLA-DR, Ron), apoptotic and neoplastic markers (e.g. BAD protein, b-myb), and novel potential markers of ovarian cancer, such as cofilin, moesin, and neuron-restrictive silencer factor (REST) protein. CONCLUSIONS: In-house production of tailored cDNA arrays, coupled to comprehensive analysis of resulting hybridization profiles, provides an accessible, reliable, and highly effective method of applying array technology to study disease processes. In the ovary, abundance of specific tumor markers, increased macrophage recruitment mediators, a late-stage angiogenesis profile, and the presence of chemoresistance-related markers distinguished normal and advanced ovarian cancer tissue samples. Detection of such parallel changes in pathway- and tissue-specific markers may prove a hallmark ready for application in reproductive disease diagnostic and therapeutic developments.

Biomarkers, Tumor↗