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BACKGROUND: The identification of cell type-specific genes and their modification under different conditions is central to our understanding of human health and disease. The stomach, a hollow organ in the upper gastrointestinal tract, provides an acidic environment that contributes to microbial defence and facilitates the activity of secreted digestive enzymes to process food and nutrients into chyme. In contrast to other sections of the gastrointestinal tract, detailed descriptions of cell type gene enrichment profiles in the stomach are absent from the major single-cell sequencing-based atlases. RESULTS: Here, we use an integrative correlation analysis method to predict human stomach cell type transcriptome signatures using unfractionated stomach RNAseq data from 359 individuals. We profile parietal, chief, gastric mucous, gastric enteroendocrine, mitotic, endothelial, fibroblast, macrophage, neutrophil, T-cell, and plasma cells, identifying over 1600 cell type-enriched genes. CONCLUSIONS: We uncover the cell type expression profile of several non-coding genes strongly associated with the progression of gastric cancer and, using a sex-based subset analysis, uncover a panel of male-only chief cell-enriched genes. This study provides a roadmap to further understand human stomach biology.
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Neoplasias Gástricas , Transcriptoma , Humanos , Masculino , Estômago , Células Epiteliais , Perfilação da Expressão GênicaRESUMO
INTRODUCTION: Rotational thromboelastometry (ROTEM® ) and thromboelastography (TEG® ) are increasingly used in the perioperative and emergency assessment of bleeding tendencies. The diagnostic value of ROTEM and TEG for von Willebrand disease (VWD) remains to be established. AIM: To investigate whether ROTEM and TEG can discriminate patients with VWD from healthy controls. METHODS: Rotational thromboelastometry and TEG whole blood coagulation profiles were compared between VWD patients (n = 100) and healthy controls (n = 89). Measures of diagnostic accuracy were calculated, including sensitivity, specificity and receiver operating characteristic (ROC) curve. RESULTS: Prolonged TEG R-time had a positive and negative predictive value (PPV, NPV) of 0.84 and 0.68 respectively. TEG clotting index (CI) had a PPV of 1.00 and an NPV of 0.60. Both R-time and CI had a high specificity and accurately discriminated VWD patients from healthy controls, with an ROC area under the curve of 0.85 and 0.99 respectively. In multivariate analysis, low FVIII levels, but not von Willebrand factor (VWF) antigen or activity, determined hypocoagulable TEG R (R2 = 0.35) and CI levels (R2 = 0.51). The ROTEM coagulation profiles of VWD patients did not differ from healthy controls. CONCLUSIONS: Thromboelastography R and CI accurately discriminated VWD patients from healthy controls, partly through the detection of low FVIII levels. The test's performance may be improved through adjustment of the test thresholds to a local reference population. Both intrinsic pathway-activated (INTEM) and tissue factor pathway-activated (EXTEM) ROTEM were of limited diagnostic value in VWD.
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Tromboelastografia/métodos , Doenças de von Willebrand/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: Embolism from unstable atheromas in the carotid bifurcation is a major cause of stroke. Here, we analysed gene expression in endarterectomies from patients with symptomatic (S) and asymptomatic (AS) carotid stenosis to identify pathways linked to plaque instability. METHODS: Microarrays were prepared from plaques (n = 127) and peripheral blood samples (n = 96) of S and AS patients. Gene set enrichment, pathway mapping and network analyses of differentially expressed genes were performed. RESULTS: These studies revealed upregulation of haemoglobin metabolism (P = 2.20E-05) and bone resorption (P = 9.63E-04) in S patients. Analysis of subgroups of patients indicated enrichment of calcification and osteoblast differentiation in S patients on statins, as well as inflammation and apoptosis in plaques removed >1 month compared to <2 weeks after symptom. By prediction profiling, a panel of 30 genes, mostly transcription factors, discriminated between plaques from S versus AS patients with 78% accuracy. By meta-analysis, common gene networks associated with atherosclerosis mapped to hypoxia, chemokines, calcification, actin cytoskeleton and extracellular matrix. A set of dysregulated genes (LMOD1, SYNPO2, PLIN2 and PPBP) previously not described in atherosclerosis were identified from microarrays and validated by quantitative PCR and immunohistochemistry. CONCLUSIONS: Our findings confirmed a central role for inflammation and proteases in plaque instability, and highlighted haemoglobin metabolism and bone resorption as important pathways. Subgroup analysis suggested prolonged inflammation following the symptoms of plaque instability and calcification as a possible stabilizing mechanism by statins. In addition, transcriptional regulation may play an important role in the determination of plaque phenotype. The results from this study will serve as a basis for further exploration of molecular signatures in carotid atherosclerosis.
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Doenças das Artérias Carótidas/genética , Transcriptoma , Idoso , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Transdução de SinaisRESUMO
The nuclear factor NF-{kappa}B (NFκB) is involved in the regulation of innate immunity and in particular, inflammatory genes. It is associated with the pathogenesis of many chronic diseases such as coronary heart disease (CHD). It is believed that individual susceptibility to CHD might be affected by differences in gene transcription and therefore gene expression in circulating cell populations such as leucocytes is of interest. The aim of this study was to investigate whether the total white blood cell population (leucocytes) could be used to study the effect of lipopolysaccharide (LPS) treatment on the expression of genes of the NFκB pathway. Gene expression of the NFκB pathway was examined in total leucocyte, monocyte and neutrophil populations. The majority of the 84 genes examined were up-regulated after treatment with LPS for 12 h in all cell populations examined. The total leucocyte population behaved in a similar manner to both neutrophils and monocytic cells, indicating that it alone could be used in studies, therefore avoiding cell separation, which is time-consuming and can result in cell activation. Furthermore, in clinical studies, it enables a larger number of patient samples to be studied simultaneously, while also reducing the amount of blood required from each. This will provide enough starting material for use with molecular techniques, such as chromatin immunoprecipitation (ChIP) and ChIP-sequencing, and allow large-scale gene expression studies of the NFκB pathway in patients with chronic and acute inflammation with established CHD.
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Leucócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Adulto , Quimiocina CCL2/genética , Fatores Estimuladores de Colônias/genética , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptor beta de Linfotoxina/genética , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.
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The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.
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BACKGROUND: Prospective studies of the epidemiology and clinical significance of low plasminogen activator inhibitor type 1 (PAI-1) activity are lacking. OBJECTIVE: To evaluate the prevalence of low PAI-1 activity in patients with a bleeding tendency in comparison with a normal population. METHODS: In 586 consecutive patients, referred because of bleeding symptoms, we added analyses of PAI-1 activity and tissue plasminogen activator complex with PAI-1 (t-PA-PAI-1) to the routine investigation, consisting of platelet count, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII, von Willebrand factor activity, and antigen. Controls were 100 blood donors and 100 age- and sex-matched healthy individuals. The latter were also evaluated regarding the previous bleeding episodes. The bleeding history was classified as clinically significant or not, and the criteria were fulfilled in 75% of the patients and 18% of the healthy controls. RESULTS: The routine laboratory investigation of the patients was negative in 57%. Low PAI-1 activity, defined as <1.0 U mL(-1), was found in 23% of the patients and in 13% and 10% of the blood donors and healthy controls, respectively (odds ratio and 95% CI, 2.04; 1.11-3.77 and 2.75; 1.39-5.42, respectively). The difference remained statistically significant after the adjustment for body mass index, use of estrogens, sex and age (odds ratio for patients vs. healthy controls 3.23; 95% CI, 1.22-8.56, P = 0.019). The distribution of the 4G/5G genotypes in the patients was not different from that of two control populations. No specific symptom predicted for low PAI-1, which did not aggravate the clinical picture in association with the other hemostatic defects. Low tPA-PAI-1 was not associated with the increased bleeding tendency. CONCLUSION: Low PAI-1 activity is common in patients with a bleeding diathesis, but it is a risk factor of minor clinical importance and not associated with specific bleeding manifestations.
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Transtornos Hemorrágicos/etiologia , Inibidor 1 de Ativador de Plasminogênio/deficiência , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Estudos de Casos e Controles , Feminino , Transtornos Hemorrágicos/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Ligação Proteica , Fatores de Risco , Fatores Sexuais , Ativador de Plasminogênio Tecidual/sangue , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.
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Alelos , Apirase/metabolismo , DNA/genética , Análise por Conglomerados , DNA/metabolismo , Genótipo , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.
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Neoplasias Encefálicas/patologia , Citomegalovirus/fisiologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/virologia , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/virologia , Transformação Celular Neoplásica/metabolismo , Intervalo Livre de Doença , Feminino , Glioblastoma/mortalidade , Glioblastoma/virologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Fenótipo , Cultura Primária de Células , Prognóstico , Esferoides Celulares/patologiaRESUMO
Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.
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Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 9 , Neoplasias Cutâneas/genética , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma de Células Escamosas/metabolismo , Análise Mutacional de DNA , Genes p53 , Humanos , Perda de Heterozigosidade , Mutação , Neoplasias Cutâneas/metabolismoRESUMO
BACKGROUND: Family history of venous thromboembolism (VTE) has been suggested to be more useful in risk assessment than thrombophilia testing. OBJECTIVES: We investigated established genetic susceptibility variants for association with VTE and evaluated a genetic risk score in isolation and combined with known trigger factors, including family history of VTE. PATIENTS/METHOD: A total of 18 single nucleotide polymorphisms (SNPs) selected from the literature were genotyped in 2835 women participating in a Swedish nationwide case-control study (the ThromboEmbolism Hormone Study [TEHS]). Association with VTE was assessed by odds ratios (ORs) with 95% confidence interval (CI) using logistic regression. Clinical and genetic predictors that contributed significantly to the fit of the logistic regression model were included in the prediction models. SNP-SNP interactions were investigated and incorporated into the models if found significant. Risk scores were evaluated by calculating the area under the receiver-operating characteristics curve (AUC). RESULTS: Seven SNPs (F5 rs6025, F2 rs1799963, ABO rs514659, FGG rs2066865, F11 rs2289252, PROC rs1799810 and KNG1 rs710446) with four SNP-SNP interactions contributed to the genetic risk score for VTE, with an AUC of 0.66 (95% CI, 0.64-0.68). After adding clinical risk factors, which included family history of VTE, the AUC reached 0.84 (95% CI, 0.82-0.85). The goodness of fit of the genetic and combined scores improved when significant SNP-SNP interaction terms were included. CONCLUSION: Prediction of VTE in high-risk individuals was more accurate when a combination of clinical and genetic predictors with SNP-SNP interactions was included in a risk score.
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Polimorfismo de Nucleotídeo Único , Trombose Venosa/genética , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Razão de Chances , Linhagem , Fenótipo , Valor Preditivo dos Testes , Curva ROC , Medição de Risco , Fatores de Risco , Fatores Sexuais , Suécia/epidemiologia , Trombose Venosa/diagnóstico , Trombose Venosa/epidemiologiaRESUMO
It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. This study was performed to elucidate age-related changes in GH-dependent effects at the level of gene expression. Through the application of complementary DNA representational difference analysis (RDA) we have identified gene products that are reduced during aging in rat liver. The expression of these genes was restored upon GH treatment. Results from reverse Northern and ribonuclease protection analysis confirmed that the RDA products were truly differentially expressed. In addition to well characterized GH-regulated genes, including CYP2C12, CYP2C13, and alpha2u-globulin, we demonstrate the differential expression of at least 11 genes previously not known to be under GH control. Several hepatic transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified. Other RDA products consisted of transcripts encoding proteins involved in ATP synthesis, detoxification of reactive oxygen species, or immune responses. This list of GH-regulated genes in the old rat may shed further light on the action and mechanism behind the positive effects of GH on, for example, body composition and the immune system that have been observed in different animal and human studies.
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Envelhecimento/metabolismo , Hormônio do Crescimento/farmacologia , Hormônio do Crescimento Humano/farmacologia , Fígado/metabolismo , Transcrição Gênica/genética , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Reações Falso-Positivas , Hipofisectomia , Hibridização In Situ , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.
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Cistina/análogos & derivados , DNA Complementar/genética , Perfilação da Expressão Gênica , Arteriosclerose/genética , Arteriosclerose/prevenção & controle , Linhagem Celular , Cistina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SoftwareRESUMO
We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.
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Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Mutação , Nucleotídeos/genética , Taq Polimerase/metabolismo , Artefatos , Sequência de Bases , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 9/genética , Análise Mutacional de DNA/métodos , Humanos , Íntrons/genética , Perda de Heterozigosidade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Moldes GenéticosRESUMO
Differential gene expression can be expected during activation and differentiation of cells as well as during pathological conditions, such as cancer. A number of strategies have been described to identify and understand isolated differentially expressed genes. The differential display methodology has rapidly become a widely used technique to identify differentially expressed mRNAs. In this chapter we described a variant of the differential display method based on solid-phase technology. The solid-phase procedure offers an attractive alternative to solution-based differential display because minute amounts of sample can be analyzed in considerably less time than previously. The employed solid support, monodisperse super paramagnetic beads, which circumvents precipitation and centrifugations steps, has also allowed for optimization of the critical enzymatic and preparative steps in the differential display methodology. We also described how bacterial expression can be used as a means to elucidate gene function. An efficient dual-expression system was presented, together with a basic concept describing how parallel expression of selected portions of cDNAs can be used for production of cDNA-encoded proteins as parts of affinity-tagged fusion proteins. The fusion proteins are suitable both for the generation of antibodies reactive to the target cDNA-encoded protein and for the subsequent affinity enrichment of such antibodies. Affinity-enriched antibodies have proved to be valuable tools in various assays, including immunoblotting and immunocytochemical staining, and can thus be used to localize the target cDNA-encoded protein to certain cells in a tissue section or even to a specific cell compartment or organelle within a cell. High-resolution localization of a cDNA-encoded protein would provide valuable information toward the understanding of protein function.
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DNA Complementar/análise , Regulação da Expressão Gênica , Animais , Anticorpos , Bactérias/genética , Sequência de Bases , Western Blotting/métodos , Clonagem Molecular/métodos , Primers do DNA , DNA Complementar/biossíntese , Eletroforese em Gel de Poliacrilamida , Humanos , Metástase Linfática , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , RNA Mensageiro/genética , Coelhos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Albumina Sérica/biossíntese , Albumina Sérica/genética , Transcrição GênicaRESUMO
Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.
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Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular , Células Espumosas , Perfilação da Expressão Gênica/métodos , Humanos , Macrófagos , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/genética , Estatística como Assunto/métodosRESUMO
A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34+CD38-). The majority of the differentially expressed fragments represented previously uncharacterized sequences.
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Antígenos CD , Expressão Gênica , Células-Tronco Hematopoéticas/química , RNA Mensageiro/análise , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos de Diferenciação/análise , Linfócitos B/química , Linfócitos B/imunologia , Proteínas de Bactérias , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Complementar/síntese química , DNA Polimerase Dirigida por DNA/metabolismo , Células-Tronco Hematopoéticas/imunologia , Humanos , Glicoproteínas de Membrana , Microesferas , N-Glicosil Hidrolases/análise , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Análise de Sequência de DNA , EstreptavidinaRESUMO
cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.
Assuntos
DNA Complementar/química , DNA de Neoplasias/química , Reação em Cadeia da Polimerase/métodos , Resinas Acrílicas/química , Animais , Biópsia , Clonagem Molecular , Neoplasias do Colo/genética , Primers do DNA/química , DNA Complementar/metabolismo , Eletroforese em Gel de Ágar , Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/farmacologia , Humanos , Fígado/efeitos dos fármacos , Masculino , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Técnica de Subtração , Extratos de TecidosRESUMO
The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13-34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.