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PubMed2005

Changes in gene expression within the nucleus accumbens and striatum following sexual experience.

Sexual experience, like repeated drug use, produces long-term changes including sensitization in the nucleus accumbens and dorsal striatum. To better understand the molecular mechanisms underlying the neuroadaptations following sexual experience, we employed a DNA microarray approach to identify genes differentially expressed between sexually experienced and sexually naive female hamsters within the nucleus accumbens and dorsal striatum. For 6 weeks, a stimulus male was placed in the home cage of one-half of the hormonally primed, ovariectomized female hamsters. On the seventh week, the two experimental groups were subdivided, with one half paired with a stimulus male. In comparison with sexually naive animals, sexually experienced hamsters receiving a stimulus male on week 7 exhibited an increase in a large number of genes. Conversely, sexually experienced female hamsters not receiving a stimulus male on week 7 exhibited a reduction in the expression of many genes. For directional changes and the categories of genes regulated by the experimental conditions, data were consistent across the nucleus accumbens and dorsal striatum. However, the specific genes exhibiting changes in expression were disparate. These experiments, among the first to profile genes regulated by female sexual behavior, will provide insight into the mechanisms by which both motivated behaviors and drugs of abuse induce long-term changes in the mesolimbic and nigrostriatal dopamine pathways.

Adaptation, Physiological↗

PubMed2005

Early antiretroviral therapy for simian immunodeficiency virus infection leads to mucosal CD4+ T-cell restoration and enhanced gene expression regulating mucosal repair and regeneration.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections lead to rapid depletion of CD4(+) T cells from gut-associated lymphoid tissue (GALT). Although the administration of antiretroviral therapy (ART) has been shown to increase CD4(+) T-cell levels in the peripheral blood in both SIV and HIV infections, its efficacy in restoring intestinal mucosal CD4(+) T cells has not been well investigated. To gain insights into the molecular mechanisms of virally induced disruptions in the mucosal immune system, we have evaluated longitudinal changes in viral burden, T-cell subsets, and mucosal gene expression profiles in SIV-infected rhesus macaques in the absence or presence of ART. Our results demonstrate a dramatic suppression of mucosal viral loads and rapid reconstitution of CD4(+) T cells in GALT in animals receiving ART that were not observed in untreated SIV-infected animals. DNA microarray-based gene expression profiling indicated that CD4(+) T-cell restoration in GALT was associated with up regulation of growth factors and genes involved in repair and regeneration of the mucosal epithelium. In contrast, untreated SIV-infected animals increased expression of lymphocyte activation and inflammatory response-associated genes and did not up regulate mucosal growth and repair associated transcription. In conclusion, these data indicate that initiating ART in primary SIV infection may lead to the restoration of the mucosal immune system through reduction of inflammation and promotion of epithelial repair in the intestinal mucosa.

Adenine↗

PubMed2007

Osteopoikilosis, short stature and mental retardation as key features of a new microdeletion syndrome on 12q14.

This report presents the detection of a heterozygous deletion at chromosome 12q14 in three unrelated patients with a similar phenotype consisting of mild mental retardation, failure to thrive in infancy, proportionate short stature and osteopoikilosis as the most characteristic features. In each case, this interstitial deletion was found using molecular karyotyping. The deletion occurred as a de novo event and varied between 3.44 and 6 megabases (Mb) in size with a 3.44 Mb common deleted region. The deleted interval was not flanked by low-copy repeats or segmental duplications. It contains 13 RefSeq genes, including LEMD3, which was previously shown to be the causal gene for osteopoikilosis. The observation of osteopoikilosis lesions should facilitate recognition of this new microdeletion syndrome among children with failure to thrive, short stature and learning disabilities.

Abnormalities, Multiple↗

PubMed2004

Soluble tumor necrosis factor-like weak inducer of apoptosis overexpression in HEK293 cells promotes tumor growth and angiogenesis in athymic nude mice.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of structurally related cytokines. TWEAK acts on responsive cells via binding to a cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production. It has also been reported that TWEAK can stimulate blood vessel formation in the rat cornea angiogenesis assay, but it is presently unknown whether this cytokine could play a role in the pathological angiogenesis associated with human diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. In the present study we investigated whether TWEAK was expressed in human tumors and whether it could promote tumor growth and angiogenesis in vivo. TWEAK mRNA expression was detected in many tumor types by cDNA array hybridization analysis, and TWEAK protein expression was confirmed in human colon cancer tissue by immunohistochemistry. As an initial approach to address whether TWEAK might act as a tumor angiogenesis factor, we established several human embryonic kidney cell lines that constitutively secrete a soluble TWEAK protein and examined their growth properties in vitro and in vivo. We found that although TWEAK-overexpressing cells do not have a growth advantage in vitro, they form larger and more highly vascularized tumors in athymic mice when compared with control, vector-transfected cells. This result suggests that the TWEAK-Fn14 signaling system may be a potential regulator of human tumorigenesis.

Animals↗

PubMed2004

Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients' plasma.

PURPOSE: The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors. EXPERIMENTAL DESIGN: Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach. RESULTS: A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients. CONCLUSIONS: Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Adolescent↗

PubMed2006

Changes in gene expression pattern following granulocyte colony-stimulating factor administration to normal stem cell sibling donors.

UNLABELLED: Granulocyte colony-stimulating factor (G-CSF) is widely used for the mobilization of hematopoietic stem cells from normal donors. The mechanism of induced mobilization has recently been elucidated. Treatment with G-CSF is considered safe; however, long-term effects are largely unknown. The aim of this study was, therefore, to determine whether it leads to significant changes in gene expression modifications. Affymetrix Gene Chip array technology was used. Samples were collected before and at various time points (up to 9 months) after mobilization. The expression levels of 122 genes were transiently modified before and after mobilization (p < 0.05). Fifty-three genes belonging to cell growth, proliferation and communication gene ontology categories were upregulated, while 69 genes were downregulated. CONCLUSION: The administration of G-CSF is associated with transient DNA and gene expression modifications in the lymphocytes of normal mobilized donors. A long-term follow-up of stem cell donors is recommended.

Down-Regulation↗

PubMed2006

Restricted localization of claudin-16 at the tight junction in the thick ascending limb of Henle's loop together with claudins 3, 4, and 10 in bovine nephrons.

Claudin-16 is one of the tight junction protein claudins and has been shown to contribute to reabsorption of divalent cations in the human kidney. In cattle, total deficiency of claudin-16 causes severe renal tubular dysplasia without aberrant metabolic changes of divalent cations, suggesting that bovine claudin-16 has some roles in renal tubule formation and paracellular transport that are somewhat different from those expected from the pathology of human disease. As the first step to clarify these roles, we examined the expression and distribution of claudin-16 and several other major claudin subtypes, claudins 1-4 and 10, in bovine renal tubular segments by immunofluorescence microscopy. Claudin-16 was exclusively distributed to the tight junction in the tubular segment positive for Tamm-Horsfall glycoprotein, the thick ascending limb (TAL) of Henle's loop, and was found colocalized with claudins 3, 4, and 10. This study also demonstrates that bovine kidneys possess segment-specific expression patterns for claudins 2-4 and 10 that are different from those reported for mice. Particularly, distribution of claudin-4 in the TAL and distal convoluted tubules was characteristic of bovine nephrons as were differences in the expression patterns of claudins 2 and 3. These findings demonstrate that the total lack of claudin-16 in the TAL segment is the sole cause of renal tubular dysplasia in cattle and suggest that the tight junctions in distinct tubular segments including the TAL have barrier functions in paracellular permeability that are different among animal species.

Amino Acid Sequence↗

PubMed2004

Dopaminergic differentiation of human embryonic stem cells.

In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)-positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K(+). When transplanted into 6-hydroxydopamine-treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2005

Death-from-cancer signatures and stem cell contribution to metastatic cancer.

Genome-wide expression profiling studies reveal a transcriptionally distinct sub-type of human solid tumors with a marked propensity toward metastatic dissemination, highly malignant clinical behavior, and poor therapy outcome in cancer patients diagnosed with the early stage carcinomas of various origins. This sub-set of tumors acquires full metastatic potential, including an emergence and seeding of potent metastasis precursor cells, early in tumor progression. Collectively, these data suggest an early involvement in development of this transcriptionally defined sub-type of human carcinomas of a highly malignant combination of mutant alleles conferring the proclivity to metastasize and/or an engagement in transformation and tumor progression of stem cells and/or early progenitor cells. Enrichment of primary tumors with normal stem cells increases the likelihood of horizontal genomic transfer (large-scale transfer of DNA and chromatin) between stem cells and cancer cells via cell fusion and/or uptake of apoptotic bodies and generation of reprogrammed somatic cell hybrids with self-renewing highly malignant phenotype.

Alleles↗

PubMed2006

Chromosomal imbalances in malignant peripheral nerve sheath tumor detected by metaphase and microarray comparative genomic hybridization.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly malignant tumors affecting adolescents and adults. There have been a few reports on chromosomal aberrations of MPNSTs; however, the tumor-specific alteration remains unknown. We characterized the genomic alterations in 8 MPNSTs and 8 schwannomas by metaphase comparative genomic hybridization (CGH). In 5 of 8 MPNSTs, microarray CGH was added for more detailed analyses. Frequent gains were identified on 3q13-26, 5p13-14, and 12q11-23 and frequent losses were at 1p31, 10p, 11q24-qter, 16, and 17. Microarray CGH revealed frequent gains of EGFR, DAB2, MSH2, KCNK12, DDX15, CDK6, and LAMA3, and losses of CDH1, GLTSCR2, EGR1, CTSB, GATA3, and SULT2A1. These genes seem to be responsible for developing MPNSTs. The concordance rate between metaphase CGH and microarray CGH was 66%. Metaphase CGH was useful for identifying chromosomal alterations before applying microarray CGH.

Adult↗

PubMed1999

In vivo gene expression profile analysis of human breast cancer progression.

The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.

Breast Neoplasms↗

PubMed2001

Simultaneous analysis of 1176 gene products in normal human aorta and abdominal aortic aneurysms using a membrane-based complementary DNA expression array.

BACKGROUND: A number of changes in gene expression have been described in abdominal aortic aneurysms (AAAs), but the spectrum of molecular alterations in this disease is unknown. The purpose of this study was to characterize the expression of approximately 1000 gene products in human AAA tissue and to compare the profile of genes expressed in AAAs with that observed in normal aorta. MATERIALS AND METHODS: Total RNA was isolated from abdominal aortic wall tissues (4 AAAs and 4 normal aortas), and array-specific [(32)P]-labeled complementary DNA (cDNA) probes were created with reverse transcription. The cDNA probes were hybridized with nylon membranes containing an array of 1176 cDNA clones (AtlasArray Human 1.2 I; Clontech, Palo Alto, Calif), and autoradiographs were scanned to identify the patterns of gene expression characteristic of each tissue type. Densitometric analysis was used to standardize the expression of individual genes to a panel of housekeeping controls, and differential gene expression was defined by a signal ratio of at least 2:1. RESULTS: One hundred forty-five (12.3%) of the 1176 genes were consistently expressed in aortic tissue. Thymosin beta-4 was the most abundant of 101 transcripts detected in both AAAs and normal aorta, whereas 44 genes exhibited differential patterns of expression (39 predominant in AAAs and 5 in normal aorta). Densitometric analysis confirmed differences in expression for 20 of these gene products between AAAs and normal aorta, with the greatest increases seen for myeloid cell nuclear differentiation antigen (31-fold), cathepsin H (30-fold), platelet-derived growth factor-A (23-fold), apolipoprotein E (13-fold), gelatinase B/matrix metalloproteinase-9 (12-fold), and interleukin-8 (11-fold). The only gene products substantially decreased in AAAs were myosin light chain kinase (39-fold) and beta-1 integrin (twofold). AAA tissues thereby exhibited a distinct pattern of gene expression reflecting chronic inflammation, extracellular matrix degradation, atherosclerosis, and smooth muscle cell depletion. CONCLUSIONS: cDNA expression arrays provide a powerful new approach to help identify the molecular mechanisms responsible for aneurysmal degeneration. Further studies will be needed to elucidate the functional and pathophysiologic significance of the individual genes that exhibit altered levels of expression in AAA tissue.

Aged↗

PubMed2004

Tissue plasminogen activator and neuroserpin are widely expressed in the human central nervous system.

Tissue plasminogen activator (tPA) is increasingly recognized to play important roles in various physiological and pathological processes in the central nervous system (CNS). Much of the data on the involvement of plasminogen activators in neurophysiology and -pathology have been derived from studies on experimental animals. We have now performed a systematic characterization of the expression of tPA and its inhibitor, neuroserpin, in normal human CNS. Brain and spinal cord samples from 30-36 anatomic locations covering all major brain regions were collected at 9 autopsies of donors with no neurological disease. Tissues were embedded in paraffin and tissue arrays were constructed. In two cases parallel samples were snap-frozen for biochemical analysis. Expression and activity profiling of tPA and neuroserpin were performed by immunohistochemistry, in situ hybridization, immunocapture and zymography assays. In the adult CNS, tPA was expressed at the mRNA and protein levels in many types of neurons, in particular in thalamus, cortex of cerebellum, pontine nuclei, neocortex, limbic system, and medulla oblongata. Interestingly, tPA was often co-expressed with its CNS inhibitor, neuroserpin. Despite overlapping expression of tPA and neuroserpin, zymography and immunocapture assays demonstrated that human neural tissue is a rich source of active tPA. Our analysis documents a detailed map of expression of tPA and its inhibitor in the human CNS and is compatible with the view that tPA is a key player in CNS physiology and pathology.

Adult↗

PubMed2002

[cDNA array approach to cytokine expression profile of aseptic loosened hip arthroplasty].

FACTS: Aseptic loosening of hip arthroplasties are the main reason for revision operations. Basic research indicates a significant relevance of the interface membrane formed between the implant an the surrounding bone. Their cellular composition and the influence of various factors on the process of aseptic loosening has attracted scientific interest. Cytokines are essential for intracellular communication. QUESTION: Is it possible to reveal differences in the expression profile of cytokines between well-fixed and failed hip arthroplasties using the cDNA array approach? AIM. Generation of a cytokine expression profile characteristic for failed hip arthroplasty. METHODS AND RESULTS: Radioactively labeled cDNA probes were synthesised from mRNA isolated from the interface membrane of six patients with aseptic loosened hip arthroplasty. Using a phosphorimager the analysis of the cDNA arrays revealed nine cytokines which were overexpressed compared with the reference tissue (Calgranulin A, Calgranulin B, IL-10, MCP-1, RANTES, TFDG1, TNFR2, RAI, THYB10). CONCLUSION: In this study four out of these nine cytokines were found to be connected with the process of aseptic loosening for the first time.

Arthroplasty, Replacement, Hip↗

PubMed2002

Protein and peptide array analysis of autoimmune disease.

Molecular cloning, sequencing of the human genome, and other major advances in biomedical research have contributed substantially to our understanding of autoimmune disease. Nevertheless, to date, such advances have failed to reveal the etiology of or yield curative therapies for autoimmune disease. New approaches are needed. Proteomics, the large-scale study of expression and function of proteins that compose our tissues and mediate disease, represents a powerful and promising strategy. We developed protein and peptide arrays to profile autoantibody responses in autoimmune disease. Protein and peptide array analysis of autoimmune samples is revealing human and pathogen proteins involved in initiation and perpetuation of autoimmunity. Proteomic determination of autoantibody profiles can be utilized for diagnosis, prognostication, and guiding tolerizing therapy for autoimmune disease.

Antigens↗

PubMed2006

Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts.

Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.

Animals↗

PubMed2002

Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research.

Over the past 5 years, microarrays have greatly facilitated large-scale analysis of gene expression levels. Although these arrays were not specifically geared to represent tissues and pathways known to be affected by diabetes, they have been used in both type 1 and type 2 diabetes research. To prepare a tool that is particularly useful in the study of type 1 diabetes, we have assembled a nonredundant set of 3,400 clones representing genes expressed in the mouse pancreas or pathways known to be affected by diabetes. We have demonstrated the usefulness of this clone set by preparing a cDNA glass microarray, the PancChip, and using it to analyze pancreatic gene expression from embryonic day 14.5 through adulthood in mice. The clone set and corresponding array are useful resources for diabetes research.

Adult↗

PubMed2003

Western array analysis of human atherosclerotic plaques: downregulation of apoptosis-linked gene 2.

OBJECTIVE: In recent years, microarray techniques have been used to characterize differences in mRNA populations between atherosclerotic plaques and normal arterial tissue. Because proteomics provide an attractive complementary approach to genomics, we used Western array technology as a global protein profiling method to identify differentially expressed proteins with potential pathobiological relevance in human atherosclerotic plaques. METHODS: Cell lysates from human carotid endarterectomy specimens and non-atherosclerotic mammary arteries were screened with monoclonal antibodies (823 in total) that were combined into unique cocktails. Hits were verified with traditional Western blotting. RESULTS: Seven proteins with a >5-fold relative expression difference were identified. One of the most apparent changes in human plaques was the downregulation of apoptosis-linked gene 2 (ALG-2), a positive mediator of apoptotic cell death. Differential expression of ALG-2 in human plaques relative to mammary arteries was not confirmed by real-time quantitative RT-PCR, suggesting post-transcriptional regulation. Uptake of aggregated LDL (agLDL) downregulated ALG-2 protein expression in THP-1 macrophages, but not in smooth muscle cells (SMCs). Transfection of THP-1 cells with ALG-2-specific small interfering RNA (siRNA) caused ALG-2 depletion and inhibited the execution phase of apoptosis (DNA fragmentation) but did not affect caspase-3 activation, annexin-V labeling and necrotic cell death. CONCLUSION: Western array screening of carotid endarterectomy specimens revealed a strong downregulation of ALG-2 protein. Because ALG-2 has pro-apoptotic potential, our results point to a novel survival mechanism against cell death in human atherosclerotic plaques.

Aged↗