RESUMO
Exosomes, the subclass of small membrane extracellular vesicles, have great diagnostic and therapeutic potential, but the lack of standardized methods for their efficient isolation and analysis limits the introduction of exosomal technologies into clinical practice. This review discusses the problems associated with the isolation of exosomes from biological fluids, as well as the principles of traditional and alternative methods of isolation. The aim of the presented review is to illustrate the variety of approaches based on the physical and biochemical properties of exosomes that can be used for exosome isolation. The advantages and disadvantages of different methods are discussed.
RESUMO
The role of infection in the beginning and progression of prostate cancer is not known to the end at the moment. Experimental data, presented in the article, are obtained by hybridization in situ and demonstrate high degree of infection of the prostate tissue by viruses with the oncogenic and oncomodulation properties HCMV, EBV, HHV8 and HPV. The authors revealed that in patients at different substages of T2 stage infection of the prostate tissue increased from T2a to T2c. The importance of these findings is discussed.
Assuntos
Infecções/virologia , Infecções por Papillomavirus/virologia , Neoplasias da Próstata/virologia , Idoso , DNA Viral/genética , DNA Viral/isolamento & purificação , Herpesviridae/isolamento & purificação , Herpesviridae/patogenicidade , Humanos , Hibridização In Situ , Infecções/genética , Infecções/patologia , Masculino , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Próstata/patologia , Próstata/virologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
The exploration of the presence of the HCMV, EBV, HHV8 and HPV-infection in the prostatic tissue of the prostate cancer patients was conducted. The methods of PCR and in situ hybridization (ISH) were used and the results were compared. Authors revealed high degree of virus infection in prostate. Using of PCR and ISH resulted in following frequencies of the viruses in prostate tissue: HCMV-87 and 77%, EBV-53 and 54%, HHV8--0 and 50%, HPV--0 and 23%. The question of the nature of discrepancies is discussed.
Assuntos
Alphapapillomavirus/isolamento & purificação , Citomegalovirus/isolamento & purificação , Infecções por Vírus Epstein-Barr/complicações , Infecções por Herpesviridae/complicações , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 8/isolamento & purificação , Infecções por Papillomavirus/complicações , Neoplasias da Próstata/virologia , Infecções Tumorais por Vírus/complicações , Idoso , Alphapapillomavirus/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/complicações , DNA Viral/isolamento & purificação , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Neoplasias da Próstata/patologiaRESUMO
Polymerase chain-reaction procedure was used to test archival samples of prostate tissue from 90 cancer patients for cytomegalovirus infection detected in 46.7%. The median Gleason score and G-index (TNM classification) were significantly higher in CMV-positive patients (8 and 4, respectively) than those in CMV-negative ones (6 and 2, respectively). A relationship between cytomegalovirus infection and poor prognosis of prostate cancer is suggested.
Assuntos
Infecções por Citomegalovirus/complicações , Neoplasias da Próstata/patologia , Neoplasias da Próstata/virologia , Idoso , Biomarcadores Tumorais/análise , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/química , Índice de Gravidade de DoençaRESUMO
Gastric and duodenal ulcer (peptic ulcer) takes the leading place among the pathology of digestive organs. According to the data of specialists from Consulting-and-Diagnostic Center N 52 among the servicemen with chronic diseases the digestive pathology constitutes 32%. Among these diseases the peptic ulcer occurs in 81% cases. Screening esophagogastroduodenoscopy conducted in 1100 servicemen without any complaints aged 22-50 has revealed duodenal ulcer in 5.4%, gastric ulcer--in 0.7% and gastroduodenitis--in 11.4%. With introduction of modern methods of diagnosis, treatment, rehabilitation and prophylaxis of peptic ulcer 98% patients with disease uncomplicated course began and completed treatment in outpatient conditions.
Assuntos
Assistência Ambulatorial , Endoscopia do Sistema Digestório , Militares , Úlcera Péptica/diagnóstico , Úlcera Péptica/terapia , Adulto , Assistência Ambulatorial/organização & administração , Humanos , Pessoa de Meia-Idade , Úlcera Péptica/prevenção & controleRESUMO
Hybridization with cDNA arrays was used to obtain expression profiles of 263 protein-tyrosine kinase (PTK), protein-tyrosine phosphatase (PTP), dual-specific phosphatase (DuSP), and other genes for the normal prostate tissue, primary prostate carcinomas (PC) of 84 patients, 7 xenografts, and 5 carcinoma cell lines. Analysis of 96 profiles revealed eight clusters of genes coexpressed in PC (coefficient of correlation r > 0.7). According to the known functions of their genes, the clusters were designated as proliferating-cell (CDC42, TOP2A, FGFR3, MYC, etc.), neoangiogenesis and blood-cell (LCK, VAV1, KDR, VEGF, MMP9, SYK, PTPRS, and FLT4), invasion-1 and invasion-2 (ADAM17, TRPM2, DUSP6, VIM, CAV1, CAV2, JAK1, PTPNS1, FYN, and PDGFB), HER2, and PSA/PSM/HER3. Basing on expression profiles of 66 genes, a molecular classification of PC was constructed and allowed discrimination between PC and cell lines or xenografts at 98.9% probability. The results suggested that, along with PSA, PSM (FOLH1), kallikrein-2, and a-2-macroglobulin, cell signaling genes EGFR, HER2, HER3, TOP2, KRT8, KRT18, VEGF, CD44, VIM, CAV1, and CAV2 may serve as diagnostic and prognostic markers in PC. The HER2, VEGF, and CD44 genes and the MMP and ADAM families were assumed to be promising targets for inhibitors of PC cell proliferation and metastasis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Monoéster Fosfórico Hidrolases/genética , Neoplasias da Próstata/genética , Proteínas Quinases/genética , Humanos , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Masculino , Família Multigênica , Monoéster Fosfórico Hidrolases/metabolismo , Próstata/fisiologia , Proteínas Quinases/metabolismo , Valores de ReferênciaRESUMO
Hybridization with cDNA arrays was used to obtain expression profiles of 214 protein-tyrosine kinase, protein-tyrosine phosphatase, dual-specific phosphatase, and other genes for kidney carcinomas (KC) and normal kidney tissues of 34 patients and for seven carcinoma cell lines. Computer analysis revealed three clusters of genes coexpressed in KC. A proliferating-cell gene cluster included MET, VIM, MYC, TOP2A, PCNA, etc. A neoangiogenesis and blood-cell gene cluster included LCK, HCK, FGR, MMP9, CSFR1, VEGF, FLT1, and KDR. A cluster corresponding to normal, differentiated kidney cells included ERBB2 (HER2) for receptor protein-tyrosine kinase, several phosphatase genes (PTPRE, PTPRB, DUSP9), and EGF. The results suggested that MET, DUSP9, PCNA, TOP2A, and VIM may serve as diagnostic and prognostic markers in KC. Tubulin and topoisomerase II were assumed to be promising targets for cell proliferation inhibitors in KC.
Assuntos
Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases/genética , Proteínas Tirosina Quinases/genética , Antígenos de Neoplasias , Biomarcadores Tumorais , Biópsia , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Humanos , Família Multigênica , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Células Tumorais Cultivadas , Vimentina/genéticaRESUMO
Embryonic stem cells (ESC) are widely employed as an experimental model owing to the similarity of molecular events occurring in their in vitro differentiation and in early embryo development. Subtraction hybridization was used to clone genes that display enhanced transcription in simple embryoid bodies (EB), which are formed on ESC culturing in suspension and are characterized by the presence of endodermal cells. The cloned sequences proved to include three new genes lacking homologs in databases. Northern analysis of the transcript tissue distribution in adult mice confirmed higher expression of these genes in differentiated cells compared with ESC.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Células-Tronco/metabolismo , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar , Embrião de Mamíferos/citologia , Camundongos , Células-Tronco/citologiaAssuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Proteínas Proto-Oncogênicas/genética , Células-Tronco/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Primers do DNA , Embrião de Mamíferos/citologia , Camundongos , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-fli-1 , Proteínas Proto-Oncogênicas c-ets , Proto-Oncogenes , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Low-dose ionizing irradiation of 16-18-day pregnant rats rapidly kills stem cells in the fetal forebrain. We have examined gamma-irradiated 17-day fetal rat brain tissue for molecular characteristics of apoptosis and changes in levels of mRNAs relevant to apoptosis. In many forebrain cells radiation elicits within 5 h nuclear condensation and fragmentation consistent with apoptosis. An electrophoretic DNA ladder indicative of internucleosomal chromatin cleavage was prominent within 3 h after irradiation. Pretreatment of pregnant rats with cycloheximide, or pretreatment of dissociated fetal brain cells in culture with actinomycin D, abolished the radiation-induced internucleosomal DNA fragmentation, demonstrating requirements for protein and RNA synthesis. Irradiation dramatically increased the level of the p53 transcription factor and the abundances of mRNAs coding for the cell-cycle inhibitor p21/Waf-1/Cip-1 and the AP-1-associated transcription factors Fos and JunB. Irradiation moderately increased the level of mRNA for the positive apoptosis regulator Bax. In contrast, irradiation reduced by 50-70% the abundances of most other mRNAs tested, including those for housekeeping proteins, p53, Jun, Myc, interleukin-1-beta-converting enzyme, and the negative apoptosis regulators Bcl-2 and Bcl-xL. These results indicate that radiation-elicited apoptosis of fetal brain cells is associated with activation of the p53 system, probable increases in AP-1 Fos/JunB heterodimers, and an increased ratio of Bax to Bcl-2 + Bcl-xL.
Assuntos
Encéfalo/efeitos da radiação , Prosencéfalo/efeitos da radiação , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos da radiação , Animais , Apoptose , Sequência de Bases , Northern Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Morte Celular , Células Cultivadas , DNA/análise , Primers do DNA , Sondas de DNA , Feminino , Feto , Raios gama , Genes fos , Genes jun , Genes myc , Genes p53 , Dados de Sequência Molecular , Gravidez , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2 , Proto-Oncogenes , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2RESUMO
Here in I summarize some cloning tips that have been revealed during construction of cDNA libraries from rat tissues and mouse stem cell lines in lambda ZAPII, and that improve the cloning efficiency. These improvements can be applied to many different vectors and cDNA cloning systems.
Assuntos
Bacteriófago lambda/genética , DNA Complementar/genética , Biblioteca Genômica , Animais , Clonagem Molecular , DNA Complementar/química , Vetores Genéticos , Camundongos , Peso Molecular , RatosRESUMO
.5 Gy prenatal neutron irradiation at the 17 day of gestation resulted in activation of gene expression in brain. Elevated level of neuronal (GAP-43, NCAM, protein kinase C, calmodulin) and protooncogene (c-foc, c-jun, c-mas) gene transcripts in brain of 2-4 weeks old rats well correlated both with brain maturation in normal animals and development of brain weight deficiency in the irradiated rats. Activation of gene expression appears to be a compensatory response of surviving brain cells to irradiation. Suppression of the gene activity in brain of 18 months old rats correlated with impairment of brain functions at later periods after prenatal irradiation.