摘要:

AbstractNormal rat hepatocytes maintained on tissue culture plastic and in serum‐supplemented medium lose their gap junctions within 12 hr and expression of their tissue‐specific functions within 24 to 72 hr. The gap junctions are lost via internalization and degradation, and the differentiated functions due to loss of synthesis and to rapid degradation of tissue‐specific mRNAs. Near normal levels of tissue‐specific mRNAs can be achieved by stabilization of the mRNAs but not by transcription (for most genes), if the cells are cultured in a serum‐free, hormonally defined medium and on substrata of tissue culture plastic, fibronectin or laminin, or on various purified collagens. The hormonally defined medium also extends the life‐span of the gap junctions to about 24 hr.Certain glycosaminoglycans, proteoglycans and anionic polysaccharides have proven to be potent inducers of gap junction expression and function, to increase abundance of tissue‐specific mRNAs, and to lower abundance of common gene mRNAs, a level of gap junctions and a pattern of gene expression similar to thatin vivo. Addition to the hormonally defined medium of 10 μg per ml of hyaluronates, dermatan sulfates, bovine lung heparan sulfate, chondroitin 4‐sulfate or chondroitin 6‐sulfate resulted in a weak response in induction of gap junctions (5 to 15% of the cells became dye and electrically coupled) and in gene expression. An intermediate response in gap junction expression (30 to 50% coupled cells) and in gene expression was observed with 50 to 100 μg per ml of heparins or hyaluronates. The most extensive coupling (70 to 100%) and the strongest responses in gene expression were seen with proteoglycans, such as dermatan sulfate proteoglycan or chondroitin sulfate proteoglycan. Treatment of cultures with several forms of carrageenans, polymers of sulfated galactose, or dextran sulfates, polymers of sulfated glucose, also gave responses in levels of gap junctions that did not always correlate with responses in gene expression. Western blots demonstrated that levels of the main intrinsic gap junction polypeptide were consistent with the degree of electrical and dye coupling.The gene expression responses (like the gap junction levels) were dependent on dosage and length of time of exposure to heparins or carrageenans and were due to an increase (tissue‐specific genes) or a decrease (common genes) in the stability of their mRNAs, and, for some liver‐specific genes, to restoration of their transcriptional signals. Tissue‐specific genes did not respond coordinately to any of the glycosaminoglycans, proteolgycans or anionic polysaccharides tested.In summary, glycosaminoglycans and proteoglycans were found to be important regulators in the synthesis and the stability of tissue‐specific mRNAs, in the stability of common gene mRNAs and in expression of gap junction protein in cultures of normal rat hepatocytes. Of especial significance is the realization that glycosaminoglycans and proteoglycans are unique among the matrix components and hormones tested to date, in that they can restore transcriptional signals for tissue‐specific mR

ISSN:0270-9139    

DOI:10.1002/hep.1840070702

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070703

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

摘要:

AbstractWe have developed the methodology for evaluating the effects of pathophysiological conditions on the molecular mechanisms of hepatic protein synthesis and fibrogenesis in baboons and man. Total RNA was extracted from percutaneous liver biopsies of five baboons who were chronically fed an ethanol‐rich liquid diet, their pair‐fed controls and from humans with a variety of liver abnormalities. Chronic alcohol administration in baboons with liver fibrosis and normal serum albumin levels increasedin vitroprotein synthesis as measured by [35S]methionine incorporation, albumin mRNA content and Type I procollagen mRNA content. There was no difference in the β‐actin (a constitutive protein) mRNA content. In humans, serum albumin levels correlated with albumin mRNA content as indicated by the intensity of dot blot hybridization and Type I procollagen mRNA levels correlated with the activity of liver fibrosis. The use of RNA‐DNA hybridization to investigate procollagen mRNA from human biopsies appears to be a valuable tool for evaluating the potential for collagen synthesis and the future course of liver disease. Besides the use of RNA‐DNA hybridization, we describe other methodologies which are useful in delineating the levels of gene expression responsible for hepatic mRNA regulation in normal liver and disease states in man. The use of molecular techniques to evaluate human liver disease provides an opportunity to develop clinically relevant information while at the same time offering the additional advantage of providing fundamental knowledge about fi

ISSN:0270-9139    

DOI:10.1002/hep.1840070704

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070705

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

摘要:

AbstractThere are numerous proteins of biological interest which cannot be obtained from natural sources in quantities sufficient for detailed biochemical and physical analysis. The limited bioavailability of these molecules has made it impossible to consider their potential utilization as either pharmacological agents and/or targets. One solution to this problem has been the development of recombinant vector systems which are designed to achieve efficient expression of cloned genes in a variety of biological systems. This paper will describe the development and application of a particular set of vectors which have been designed to achieve efficient expression of essentially any gene coding sequence inEscherichia coli. The system utilizes efficient phage‐derived transcriptional and translational regulatory signals and provides a strong regulatable promoter, an antitermination mechanism to ensure efficient transcription across any gene insert, high stability and, when appropriate, efficient translation initiation information. In addition, a wide variety of host strains have been developed in order to help control, stabilize and maximize expression of various cloned genes. The system has now been used to express efficiently more than 75 different prokaryotic and eukaryotic gene products. The application of this system to the expression and characterization of several oncogene products will be describe

ISSN:0270-9139    

DOI:10.1002/hep.1840070706

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070707

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070708

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070709

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070710

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY

ISSN:0270-9139    

DOI:10.1002/hep.1840070711

出版商:W.B. Saunders    

年代:1987

数据来源: WILEY