RESUMO
The silicon isotope composition of chert has recently been used to study the historic evolution of the global ocean. It has been suggested that Precambrian cherts have much higher δ30Si values than Phanerozoic cherts do and that the former show an increasing trend from 3.5 to 0.85 Ga, reflecting a decrease in ocean temperatures. However, cherts have various origins, and their isotopic compositions might be reset by metamorphic fluid circulation; thus, different types of cherts should be distinguished. Here, we present a new set of δ30Si data for cherts from early and middle Proterozoic carbonate rocks from Northern China. We found that cherts of 1.355-1.325 Ga show a peak range of 2.2-3.9. Based on these results, we propose that from the Archean to the middle Proterozoic, there was a drastic decrease in silicon content and an increase in the δ30Si value in ocean water due to a temperature decrease and biological activity increase. After that period, the silicon content of the ocean was limited to a low level by a high degree of biological absorption, and their δ30Si values varied in a small range around a significantly lower value.
RESUMO
We applied the technique of mRNA differential display to normal liver tissue and hepatoma cell line Hep3B. One of the isolated cDNA clones was expressed in human normal liver tissue but not in the human hepatocarcinoma cell line. Northern Blot analysis confirmed that high level of mRNA was expressed in human normal liver tissue but the level was decreased in non-cancerous liver tissue from hepatoma patients. Low level or no expression was observed in human hepatoma tissue. One of these transcripts was about 1.8 kb in length. Southern Blot analysis showed that it was a single copy gene. We obtained a full length cDNA clone of 2,395 bp by screening human liver 5'-stretch plus cDNA library. Nucleotide sequence indicated that this clone was highly homologous to aryl-dialkyl-phosphatase and possessed two polymorphic sites. Aryl-dialkyl-phosphatase which has a prominent role in the metabolism of several toxic, synthetic compounds, may be potentially related to human hepatocarcinoma susceptibility. The biological significance of its differential expression in normal versus malignant tissue is discussed.
Assuntos
Carcinoma Hepatocelular/química , Esterases/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Neoplasias Hepáticas/genética , Fígado/química , RNA Mensageiro/análise , Sequência de Aminoácidos , Arildialquilfosfatase , Sequência de Bases , Clonagem Molecular/métodos , DNA Complementar/genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes , Humanos , Neoplasias Hepáticas/química , Dados de Sequência Molecular , RNA Neoplásico/análise , Células Tumorais CultivadasRESUMO
Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-beta-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with the relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.
Assuntos
Técnicas de Transferência de Genes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva , Bovinos , Expressão Gênica , Marcação de Genes , Genes Reporter , Heparina/farmacologia , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Peptídeos , Protaminas , Receptores de Fatores de Crescimento do Endotélio Vascular , Células Tumorais Cultivadas , beta-Galactosidase/genéticaRESUMO
Using the human liver cancer DNA transfected NIH/3T3 cell line, the human N-ras oncogene and the over expression of the oncoprotein P21ras was demonstrated, BALB/C mice were immunized. The spleen cells from the immunized mice were fused with SP2/0 myeloma cells. After the HAT medium selection and screening, two hybridoma cell lines, SCI-Oncogema 1 and 2, were established. In the immunoprecipitation test, the molecular weight of the protein reacting to Oncogema 1 was 21,000. This M.W 21,000 protein possessed the capability to bind with GTP, i.e. the character of P21ras. These data indicate that the Oncogema 1 is the monoclonal antibody against P21ras. Using Oncogema 1, specimens from 6 liver cancer patients were studied by immunopathology. With ABC stain, it was observed that the malignant cells in all the samples showed dark staining; the P21ras revealed over expression. Although the staining was heterogeneous, it implied that the ras oncogene was involved in the carcinogenesis of these six samples. No over expression was seen in the normal liver cells even in those around the cancerous lesion. However, dysplastic cells were moderately stained which means that the ras oncogene was activated and P21ras over expressed in these cells. The results suggest that the ras oncogene and P21ras play an important role in the early stage of liver cancer carcinogenesis.
Assuntos
Neoplasias Hepáticas/genética , Oncogenes , Proteínas Proto-Oncogênicas/análise , Animais , Anticorpos Monoclonais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Transfection assay of NIH 3T3 cells was performed with DNAs isolated from ten human PHC (primary hepatic cancer) specimens and a hepatoma 7402 cell line. Positive results were obtained in 7402 and in six out of ten PHC DNAs. Human N-ras gene was identified in transfectants from 7402 DNA and transformed cells from three PHC DNA samples, which had passed more than two cycles of transfection. The expression of N-ras was also remarkably enhanced in six out of nine poly(A)+RNA samples isolated from PHC tissues. P21 synthesis was elevated in 7402 cells as well as in transfectants derived from 7402 cells and PHC DNA. In analysis of PHC DNA, rearrangement and amplification of N-ras gene was observed in two PHC samples. The discrepancy of results of the transfection assay and mRNA expression was discussed. Furthermore, c-myc was also highly expressed in most PHC tissues. It implied that the cooperating activity of N-ras and c-myc might be responsible for the malignant phenotypic alteration in some or most cases in human primary liver cancer.
Assuntos
Neoplasias Hepáticas/genética , Oncogenes , Autorradiografia , Carcinoma Hepatocelular/genética , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Amplificação de Genes , Regulação da Expressão Gênica , Humanos , Hibridização de Ácido Nucleico , Fenótipo , Poli A/metabolismo , RNA/metabolismo , RNA Mensageiro , TransfecçãoRESUMO
OBJECTIVE: To investigate molecular events associated with thymus dysfunction in the spontaneously hypertensive rat (SHR), we analysed the difference in gene expression of the thymus between SHR and Wistar-Kyoto (WKY) rats. METHODS: cDNA representational difference analysis (cDNA-RDA) was applied to compare the gene expression in the thymus between SHR and WKY. The difference products of cDNA-RDA were cloned into pBluescript SK (+) for differential screening. The positive clones were sequenced, and the sequences were analysed using the BLAST program for the homology search. Northern blot analysis was used to confirm the results of differential screening. RESULTS: The results of differential screening showed that eight genes were over-expressed in the thymus of SHR. Comparison of eight sequences against the databases identified four known and four novel genes. The known genes included cathepsin B, AT1-46, cytochrome C oxidase subunit I and metastasis suppressor homolog (KAI1). Two of the novel genes are members of immunoglobulin superfamily genes, and the rest of the novel genes have no significant homology to non-redundant GenBank + EMBL + DDBJ + PDB. By Northern blot hybridization, cathepsin B and clone #7 (GenBank accession #AF106623) genes were confirmed to be overexpressed in the SHR thymus. CONCLUSION: cDNA-RDA combined with differential screening can be used to detect easily the differentially regulated genes in two comparable tissues. The eight isolated genes are good clues toward understanding the development and function of SHR thymus.
Assuntos
DNA Complementar/genética , Hipertensão/genética , Timo/metabolismo , Regulação para Cima/genética , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The 5' region of the mouse gamma-glutamyltransferase (gamma GT; EC 2.3.2.2) gene has been cloned and analyzed. This analysis, combined with sequence information obtained from gamma GT cDNA clones, indicates that in mouse a single gamma GT gene codes for six different mRNAs that differ in their 5' sequences. Analysis of steady-state levels of gamma GT RNA reveals different expression patterns for these RNAs in different organs. The six different 5' sequences are widely separated within a 10-kb region and three of them show 75-86% identify with the three known rat gamma GT cDNAs. Although the heterogeneity of the 5' ends of gamma GT RNAs may be explained in part by alternative splicing, it is likely that multiple promoters are involved in their generation.
Assuntos
RNA Mensageiro/química , gama-Glutamiltransferase/genética , Animais , Sequência de Bases , Clonagem Molecular , Rim/química , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Especificidade de Órgãos , Regiões Promotoras Genéticas , RNA Mensageiro/análiseRESUMO
Mouse gamma-glutamyl transpeptidase (gamma GT) is encoded by a single copy gene with at least five and probably six different promoters directing the transcription of six types of gamma GT RNAs. In mouse small intestine, only Type I, V, and VI gamma GT RNAs are detected, and ribonuclease protection assays reveal that Type VI represents more than 90% of gamma GT RNA. To investigate the structure of intestinal gamma GT RNA in greater detail, we cloned and sequenced mouse intestinal gamma GT cDNAs. Seven of eight informative clones were Type VI and consisted of Type VI unique exons, VIa and VIb (as described previously by us) (Rajagopalan, S., Wan, D.-F., Habib, G. M., Sepulveda, A. R., McLeod, M. R., Lebovitz, R. M., and Lieberman, M. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6179-6183) as well as common 3' sequences. Exon VIb contains two alternative splice acceptors, one previously identified by us and the other 17 bases 5' of this site. Another clone contained a previously unidentified gamma GT mRNA designated as Type VII. Type VII consists of a unique 5' exon which is 315 base pairs upstream of the exon VIa splice donor site and is spliced to exon VIb. Regulation of gamma GT expression in the small intestine is complex and involves at least three previously described promoters, alternative splicing, and a previously undescribed exonic sequence (Type VII RNA) 5' of promoter VI.
Assuntos
Regulação Enzimológica da Expressão Gênica , Intestino Delgado/enzimologia , RNA/análise , gama-Glutamiltransferase/genética , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.
Assuntos
Linfócitos B/imunologia , Epitopos/análise , Vírus da Hepatite B/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Vírus da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Plasmídeos , Proteínas Recombinantes de Fusão/imunologia , Mapeamento por Restrição , Transativadores/síntese química , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
gamma-Glutamyl transpeptidase is a key enzyme in glutathione (GSH) salvage, metabolism of endogenous mediators such as leukotrienes and prostaglandins, detoxification of xenobiotics including environmentally important compounds and carcinogens, and cellular processes dependent on the oxidation/reduction of glutathione. The enzyme is widely distributed, and these functions often occur in separate tissues and in response to different stimuli. Evidence indicates that gamma-glutamyl transpeptidase plays a direct role in some hepatic and renal responses to injury. In the mouse gamma-glutamyl transpeptidase is a single copy gene expressed from at least seven promoters, and many of the transcribed gamma-glutamyl transpeptidase RNAs are restricted in their expression. Studies that combine analyses of cellular processes with a knowledge of gene structure and expression hold promise for unravelling how these two different levels of function are integrated.
Assuntos
Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/fisiologia , Animais , Humanos , gama-Glutamiltransferase/químicaRESUMO
We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.
Assuntos
Regiões Promotoras Genéticas , RNA Mensageiro/análise , Transcrição Gênica , gama-Glutamiltransferase/genética , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Feto/enzimologia , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , DNA de Neoplasias/genética , Neoplasias Hepáticas/genética , Oncogenes , Animais , Linhagem Celular , Transformação Celular Neoplásica/análise , HumanosRESUMO
We have identified LASS2, a previously unknown human homologue of the yeast longevity assurance gene LAG1. The LASS2 transcript is highly expressed in liver and kidney, which is very different from the expression of the previously identified human LAG1 homologue LAG1Hs-1. Radiation hybrid mapping studies indicated that LASS2 is located on chromosome 1q11. Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed that the LASS2 protein interacts with several membrane-associated receptors or transporters. Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatoma cells in vitro, which suggests that this gene may be involved in the regulation of cell growth.