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E Lai

Publications and source records attributed to E Lai.

At least 109 records · Page 6Linked to original sources
PubMed1988

The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites.

We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.

Animals↗
PubMed1987

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin.

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.

Alleles↗
PubMed1987

Genomic organization of the mouse T-cell receptor beta-chain gene family.

We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments.

Animals↗
PubMed1986

Analysis of a human V beta gene subfamily.

We have isolated and sequenced five germline V beta gene segments that are homologous to the V region of the YT35 cDNA encoding the beta chain of the T cell antigen receptor from the tumor MOLT-3. One of these gene segments is identical to the YT35 V segment, and therefore is the corresponding germline V beta gene segment encoding the YT35 cDNA. The other four V beta members exhibit 77-98% homology to the YT35 V gene segment. Two of these V beta gene segments are pseudogenes. Analyses of the coding region sequences reveal that, although the V beta segments are very diverse, they are mutating at a rate comparable to that observed in most eukaryotic genes. Analyses of the genomic clones show that the spacing distance between germline V beta gene segments ranges from 3 kb to greater than 30 kb, and the entire V beta 8 subfamily appears to be linked by a total of no more than 110 kb of DNA.

Base Sequence↗
PubMed1986

Human T-cell receptor genes: organization, diversity, and polymorphism.

The analysis of 27 human beta cDNA clones has revealed striking insights as to the germ line, combinatorial, and somatic mechanisms that operate to diversify V beta gene segments. It appears that the repertoire of human V beta genes is limited to 60 or so functional gene segments. Deletional mapping together with the analysis of large DNA fragments by field inversion gel electrophoresis and the isolation of cosmid clones should permit us to map the entire human V beta gene family. The polymorphisms that are present in these V beta gene segments will facilitate a search for interesting disease associations. A detailed comparison of the mouse and human V beta loci should permit us to come to understand in more detail the evolutionary mechanisms that have created these fascinating gene families.

Base Sequence↗
PubMed1986

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Animals↗
PubMed1985

Genetic and nongenetic control of the immune response of mice to a synthetic glycolipid, stearylisomaltotetraose.

Evidence for genetic control of antibody response to stearylisomaltotetraose (ST-IM4), a chemically defined synthetic glycolipid, was studied in various mouse strains. Anti-glycolipid antibodies were induced by repeated injections of ST-IM4 in complete Freund's adjuvant. C57BL and CBA/J mice were found to be good responders, while A/J, SJL/J, and AKR/J mice were poor responders. Responsiveness is independent of the H-2 genotype since AKR/J and CBA/J mice share the same H-2 locus. In addition, B10.A and B10.PL mice developed antibody levels similar to those of C57BL. The immunoglobulin heavy-chain locus (IgCH) was also found not to control antibody levels to ST-IM4 since C57BL and SJL/J mice share the same IgCH allotype. In C58/J mice, wide variation in response was observed among individual mice. Study of the response to ST-IM4 in 12 breeder pairs from different family lines of the C58/J colony also indicated that the regulation of the immune response to ST-IM4 is apparently more complex than can be explained by single gene control. C58 mice, unlike many other strains, had very low preimmune titers.

Animals↗
PubMed1985

Studies on the suanzaorentang in the treatment of anxiety.

We studied the anxiolytic and psychomotor effects of the ancient Chinese formula of Suanzaorentang, and found that it improved mood, decreased the accompanying sympathetic symptoms and improved psychomotor performance during the day.

Anti-Anxiety Agents↗
PubMed1985

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: production and characterization of mouse hybridoma antibodies specific for stearyl-isomaltosyl oligosaccharides.

Twelve C57BL/6J hybridoma clones, 9, 2 and 1 from mice immunized with stearyl-isomaltotetraose, stearyl-isomaltopentaose and stearyl-isomaltohexaose respectively were characterized. Seven produced IgA and 5 IgM. The specificities and sizes of their combining sites were determined by quantitative precipitin and precipitin inhibition assays. All 12 hybridoma antibodies precipitated with alpha 1----6 dextran B512 and linear dextran LD7, indicating that they recognize an internal -Glc alpha 1----6Glc alpha 1----6Glc- determinant. This in contrast with the results with rabbit antisera obtained in response to the same immunogen which recognize the non-reducing terminal determinant Glc alpha 1----6Glc alpha 1----6Glc-. Of the 12 hybridoma antibodies, 1 has an antibody combining site complementary to 4 alpha 1----6-linked glucoses while others have combining sites complementary to isomaltohexaose or isomaltoheptaose. The large combining-site sizes found in C57BL/6 hybridoma clones may be related to the pre-existing clonal repertoire in this strain. Binding constants of monomers of these antibodies for dextran B512 and isomaltoheptaose determined by affinity electrophoresis range from 1.4 X 10(3) to 4.6 X 10(5) ml/g and from 1.2 X 10(3) to 3.5 X 10(4) M-1 respectively, which is consistent with previous studies in the anti-dextran B512 system. The use of synthetic glycolipids as antigens enables us to study the gene control of antibody responses to glycolipids and to investigate the combining-site specificities of antibodies to a single antigenic determinant. Results so far show that all 12 hybridoma proteins are different despite the simplicity of the antigens. The findings provide further insight into the specificity of antibody combining sites.

Animals↗
PubMed1982

Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes.

The subtype of the beta-adrenergic receptor expressed in 3T3-L1 preadipocytes and adipocytes differentiated with dexamethasone and methylisobutylxanthine was determined by comparing the affinity of the receptors for epinephrine, norepinephrine, and beta-1 and beta-2 selective antagonist, 8-fold more avidly than adipocyte receptors. In contrast, adipocyte beta-receptors had a 10-fold higher affinity for epinephrine than for norepinephrine and complexed the beta-2 selective agonist zinterol with a 20-fold higher affinity than preadipocyte receptors. Hofstee plots and computer analyses of the binding data revealed that the populations of beta-1 receptors in preadipocytes and beta-2 receptors in adipocytes were nearly homogeneous. Preliminary characterizations of the beta-receptor phenotype in (nondifferentiating) 3T3-C2 cells treated with dexamethasone and methylisobutylxanthine and 3T3-422A adipocytes differentiated with insulin indicated that the expression of beta-2 receptors was not correlated with differentiation, but rather with exposure of the cells to dexamethasone and methylisobutylxanthine. The regulator of beta-receptor subtype was identified as the glucocorticoid analog, dexamethasone, by employing 3T3-L1 adipocytes which were stimulated to differentiate with methylisobutylxanthine and insulin. Detailed binding studies showed that under these conditions the adipocyte receptors retain beta-1 character. Subsequent treatment with 0.5 microM dexamethasone promoted the loss of beta-1 receptors, the appearance of beta-2 receptors, and a net 2- to 3-fold increase in the number of beta-receptors. Dexamethasone effected a complete switch from beta-1 to beta-2 subtype at concentrations as low as 2.5 nM while other steroids were ineffective below a concentration of 10 microM.

Adipose Tissue↗
PubMed1977

Acquisition of increased hormone sensitivity during in vitro adipocyte development.

Murine 3T3-L1 fibroblasts enter a differentiation program subsequent to prolonged maintenance in the confluent state and develop into adipocytes. The hormone sensitivity of adenylate cyclase and the physiological responsiveness to insulin were compared in 3T3-L1 preadipocytes and adipocytes. The following observations, comprising several distinct categories of hormone responsiveness, were made. (a) (2.5 micronM) isoproterenol stimulated adenylate cyclase 15-fold in adipocyte homogenates, but only 2.5-fold in preadipocyte preparations, suggesting a considerable magnification in beta-adrenergic responsiveness during development. (b) A totally new control element, adrenocorticotropic hormone responsiveness, was incorporated into the adenylate cyclase system of the adipocytes. (c) Sensitivity to prostaglandin E1 was observed in both preadipocytes and adipocytes, but no change in responsiveness could be detected in the differentiated cells. (d) Glucagon-sensitive adenylate cyclase could not be detected in either preadipocytes or adipocytes. (e) Both preadipocytes and adipocytes possess considerable insulin binding activity, but near physiological levels of insulin stimulate the conversion of glucose to CO2 and lipid only in the differentiated cells.

Adenylyl Cyclases↗
PubMedDate not supplied

Pulsed field gel electrophoresis: studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system.

We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions. We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs. We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination. The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters.

Autoanalysis↗
PubMedDate not supplied

Separation of large DNA molecules with high voltage pulsed field gel electrophoresis.

We have developed two high voltage (3 kV and 10 kV) high speed pulsed field gel electrophoresis systems for the separation of DNA as large as 460 kbp. These systems enable us to combine the rapid speed of high voltages and the separation power of pulsed field electrophoresis to achieve high resolution and short run durations. We found that large DNA fragments can be separated at voltage gradients much higher than commonly used. Yeast chromosomes as large as 460 kbp can be separated in 4 h at 20 V/cm and 1-50 kbp DNA can be rapidly separated in 30 min at 55 V/cm. This is 25 times faster in mobility for the separation of relatively small DNA fragments (< 50 kbp). We have also found an inverse relationship between the voltage applied and the size separation limit at that particular voltage gradient (55 V/cm limits the separation to 50 kbp while 20 V/cm can separate up to 460 kbp). Depending on the size range, DNA can be separated 8- to 25-fold faster and with better resolution than existing electrophoretic systems.

Adenoviruses, Human↗
PubMedDate not supplied

Hepatitis B viral nucleotide sequences in non-A, non-B or hepatitis B virus-related chronic liver disease.

The presence of serological markers of hepatitis B virus (HBV) infection and of hepatocellular HBV DNA were investigated in 19 HBsAg-negative patients with clinically and histologically significant chronic liver disease. Four cases negative for antibodies to HBsAg (anti-HBs), to the core antigen (anti-HBc), and to the e antigen (anti-HBe) were classified as non-A, non-B hepatitis. The remainder, positive for one or more of the three antibodies, were classified as hepatitis B. Histologic diagnosis was chronic active hepatitis in five, chronic persistent hepatitis in 11, micronodular cirrhosis in two, and fatty liver in one patient. The DNA extracted from limited amounts of liver biopsies, without cleavage by restriction endonucleases, was analyzed by the Southern blot technique for the presence of episomal HBV DNA. Autoradiographs showed a single band of less than 4.0 kilobase (kb) corresponding to the monomeric form of HBV DNA in five patients, several bands of larger forms (4.0 to 18.0 kb) in three patients, both the monomeric and the larger forms in eight patients, and no HBV DNA in three patients. While HBV DNA was detected in the hepatocellular DNA of six patients who underwent splenectomy, hybridization was negative with the DNA extracted from their spleens. The episomal viral DNA larger than 4.0 kb may represent concatemeric forms or free oligomers which could not be distinguished from rearranged and/or integrated viral DNA in the limited analyses of the hepatocellular DNA hydrolyzed with HindIII or EcoRI. Our observations suggest the presence of HBV-like agents in the liver of serologically HBsAg-negative patients with chronic liver disease.

Adolescent↗
PubMedDate not supplied

Hepatic expression of hepatitis B and delta virus antigens in patients with acute and chronic hepatitis.

Delta antigen (delta-Ag), hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) were examined by immunofluorescence and immunoperoxidase staining in 106 deparaffinized liver biopsy samples from HBsAg-positive patients with acute and chronic hepatitis. The delta-Ag was present in 15 cases (14%), with nuclear positivity varying greatly in intensity and prevalence. Patients with chronic hepatitis associated with delta infection had a histological picture characterized by foci of intralobular inflammation, many apoptotic bodies and shrunken hepatocytes with no satellite signs of inflammation. The histological pattern of delta antigen-positive acute hepatitis was characterized by the presence of a large number of intralobular apoptotic bodies. The inflammatory reaction mainly involved the portal tracts with piecemeal necrosis, but without collagen production. In the same cases the pattern of expression of HBsAg and HBcAg was unusual: in three cases HBcAg and HBsAg were concomitantly present, whereas in one case none of the hepatitis B virus markers was detectable.

Acute Disease↗