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J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 91 records · Page 5Linked to original sources
PubMed1991

Identification of sequences mediating interleukin-6 induction of a rat T kininogen gene.

Interleukin-6 is a pleiotropic cytokine that has a major role in the coordination of the hepatic acute phase response. In order to more fully understand this role, we have examined the interleukin-6 induction of T kininogen, a cysteine protease inhibitor and a major acute phase reactant in the rat. Using deletional analysis and site-directed mutagenesis of T kininogen-chloramphenicol acetyltransferase fusion constructs transfected into HepG2 hepatoma cells, we have identified two similar interleukin-6 response elements within 250 base pairs of the transcription start site. These two response elements are functionally interdependent. The sequences of these two elements match the consensus sequence for the previously described Type B interleukin-6 response element. Interleukin-6 signal transduction via two Type B elements has not been observed previously in vivo. A DNA fragment encompassing these response elements forms the same protein complex with nuclear extracts from both untreated and interleukin-6-treated cells.

Animals↗
PubMed1991

Transcription factor OTF-1 interacts with two distinct DNA elements in the A gamma-globin gene promoter.

A DNA region (site II) in the promoter of the human A gamma-globin gene (-182 to -168) is involved in transcriptional regulation. At least two nuclear proteins bind to this region: the erythroid-specific factor NF-E1/GF-1 and another factor present in many cell lines. In the present study, we demonstrate that the ubiquitous factor binding to site II has immunological identity with the octamer transcription factor OTF-1, which has been implicated in the regulation of expression of genes such as histone H2b and small nuclear RNA. In addition, we show that OTF-1 binds to site I (-291 to -267), a purine-rich region upstream of site II. Interestingly, OTF-1 binds to sites I and II with equal affinity. This was unexpected since the 14 bp site I binding site AAGAATAAATTAGA (-291 to -278), determined by methylation interference, does not show obvious similarities to the canonical octamer binding site for OTF-1 in site II (ATGCAAAT). Interaction of OTF-1 with functionally active binding sites in the gamma-globin promoter suggests that this factor has a role in gamma-globin transcription.

Base Sequence↗
PubMed1991

Developmental and tissue-specific expression of rat T-kininogen.

The ontogeny of T-Kininogen expression in the rat liver was examined. Levels approximately 6 fold higher than seen in the adult liver are present during the perinatal period. Elevated levels are also seen in the maternal liver, beginning 5 days prior to parturition. The timing of induction in the fetal and maternal liver is distinct, suggesting independent regulation. While T-Kininogen mRNA is mainly synthesized in the liver, low but significant expression is seen in other tissues, including lung and kidney.

Aging↗
PubMed1990

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit.

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.

Animals↗
PubMed1990

Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes.

The Na,K-ATPase alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes exhibit a complex pattern of expression during heart development. To identify possible molecular signals that regulate the differential expression of these genes, isolated neonatal rat myocardial and non-myocardial cells were cultured in chemically defined medium and the responses of the multiple Na,K-ATPase subunit mRNAs to various hormones were tested. Myocardiocytes in control cultures express primarily alpha 1 and beta mRNAs. Triiodothyronine (T3) induced the expression of alpha 2, alpha 3, and beta mRNAs without influencing alpha 1 mRNA levels. Dexamethasone (DEX) treatment similarly induced alpha 2 mRNA levels, but the abundance of the other subunit transcripts remained unaltered. T3 and DEX together caused increases in alpha 2 and beta mRNA, increments similar to that observed with T3 alone. However, DEX specifically repressed the induction of alpha 3 mRNA by T3. Both hormones stimulated corresponding changes in the sarcolemma concentration of these Na,K-ATPase isozymes. Addition of norepinephrine to the cultures had little appreciable effect on expression of the alpha isoform and beta mRNAs. Although characterized less extensively, control cultures of non-myocardiocytes expressed alpha 1, alpha 3, and beta mRNAs, of which only the beta mRNA was stimulated by T3. These data indicate that thyroid and glucocorticoid hormones differentially regulate the expression of multiple alpha isoform and beta subunit mRNAs of Na,K-ATPase in cardiocytes in vitro and, therefore, may also be important physiological modulators in vivo.

Animals↗
PubMed1990

Characterization of the 5'-flanking region of the human and rat Na,K-ATPase alpha 3 gene.

Genomic clones containing the 5'-flanking region and exon 1 of the human and rat Na,K-ATPase alpha 3 isoform gene have been isolated and characterized. The nucleotide sequences of 1.6 kb of the rat gene and 2.8 kb of the human gene in the 5'-flanking region were determined. Mapping of transcription initiation sites by primer extension and S1 nuclease protection analyses indicates that transcription is initiated in the same region in both genes although the rat gene has a greater number of initiation sites. Neither gene has a canonical TATA box, having instead a ATAT sequence preceding the transcription initiation sites. There is a perfect CCAAT sequence, in the reverse orientation, approximately 30 bp upstream of the potential TATA box in both genes. We have identified potential binding sites for transcription factors Sp-1, AP-1, AP-2, and AP-4, as well as for glucocorticoid and thyroid hormone receptors in the 5'-flanking regions. These are conserved in both human and rat alpha 3 isoform genes.

Animals↗
PubMed1990

The human Na, K-ATPase alpha 1 gene: characterization of the 5'-flanking region and identification of a restriction fragment length polymorphism.

We have determined the sequence of the 5'-flanking region and first three exons of the human Na,K-ATPase alpha 1 gene, ATP1A1. Primer extension and S1 nuclease protection analyses of RNA from human kidney, brain, and skeletal muscle indicate that transcription initiates 273 nucleotides upstream of the translation start site. The promoter region contains a potential TATA box at position -27 relative to the transcription initiation site; however, no CCAAT sequence is observed. The 5'-untranslated and 5'-flanking regions are G + C rich. Five sequence elements exhibiting similarity to binding sites for the transcription factor Sp1 are located within the 5'-flanking region. This region also contains potential binding sites for the transcription factors AP-1, AP-2, AP-3, and NF-1, as well as a site which exhibits perfect identity to an 8-bp sequence element important for calcium induction. A comparison of the 5'-flanking region of the alpha 1 and alpha 2 genes reveals differences in potential transcription factor and hormone receptor binding sites which may be important in mediating the tissue- and developmental stage-specific expression of these genes. We have also identified an intragenic DNA probe which detects a restriction fragment length polymorphism at the alpha 1 locus. This marker should facilitate genetic linkage studies designed to evaluate the role of the sodium pump in human disease.

Base Sequence↗
PubMed1990

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Amino Acid Sequence↗
PubMed1990

Differential regulation of rat T-kininogen by tumor necrosis factor and interleukin-6.

Rat T-kininogen (T-KG), a cysteine protease inhibitor, is an acute phase reactant which is induced to high levels in response to inflammation. Both hormones and cytokines participate in this regulation. To investigate the cis-acting elements responsible for the induction of gene expression, various 5'-fragments of the rat T-KG gene were fused to a chloramphenicol acetyltransferase marker gene. These constructs were transfected into a rat hepatoma cell line which was then treated with tumor necrosis factor or interleukin-6 or both cytokines. Expression of the chloramphenicol acetyltransferase gene was induced with interleukin-6 treatment, but suppressed by tumor necrosis factor. The 5'-region of the T-KG gene responsible for conferring both of these effects was localized between nucleotides -404 to -210 upstream of the transcription start site. Fragments containing this region were found to be effective in either orientation, and could also regulate a heterologous promoter.

Acute-Phase Proteins↗
PubMed1989

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase.

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.

Amino Acid Sequence↗
PubMed1989

Characterization of the human Na,K-ATPase alpha 2 gene and identification of intragenic restriction fragment length polymorphisms.

We have determined the structure of the gene that encodes the alpha 2 isoform of the human Na,K-ATPase. The gene contains 23 exons and spans approximately 25 kilobases. The amino acid sequence of the human alpha 2 isoform deduced from the genomic sequence exhibits 99% identity to the rat alpha 2 isoform. One of the nine amino acid differences between the human and rat sequences occurs at an amino acid position which is known to be involved in species differences in sensitivity of the alpha 1 isoform to cardiac glycosides. Approximately 1500 base pairs of sequence flanking the 5' end of the alpha 2 gene have been determined. This region contains numerous potential AP-1, AP-2, and NF-1-binding sites, a potential Sp1 recognition site, and several sequences that are similar to the glucocorticoid receptor-binding site. The transcription start site was mapped by primer extension and S1 nuclease protection analyses of RNA from human brain, skeletal muscle, and heart. Multiple transcription initiation sites are clustered between residues -104 to -99 relative to the translation initiation codon. A potential TATA box is located 29 base pairs upstream of the first transcription initiation site. Immediately 5' to the apparent TATA box is a 35-base pair polypurine.polypyrimidine tract containing an imperfect mirror repeat which resembles sequences that form triple-stranded structures. Two intragenic DNA probes which detect restriction fragment length polymorphisms associated with the alpha 2 gene have been identified. These probes will be useful in genetic linkage analyses designed to define the possible role of the Na,K-ATPase in certain hereditary disorders.

Amino Acid Sequence↗
PubMed1989

Primary structure of a gene encoding rat T-kininogen.

A gene encoding the acute-phase reactant, T-kininogen, has been isolated from a rat genomic library. The nucleotide sequence of all exons as well as of a large portion of the introns was determined. The gene, T-KG, is approximately 24 kb in length and contains 11 exons. The promoter appears to be rather unusual in that there are no consensus CCAAT or TATAA boxes or SP1-binding sites. An A-rich region present upstream of the transcription start point may function as a TATAA-equivalent sequence. In addition, several potential glucocorticoid regulatory elements and a sequence similar to the AP-1 binding site are present in the 5' region of the gene. The intron/exon boundaries in this T-KG gene are identical to those seen in the human K-kininogen gene. This is consistent with the inclusion of T-KG in the cystatin supergene family of cysteine protease inhibitors.

Amino Acid Sequence↗
PubMed1989

Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator.

Base Sequence↗
PubMed1989

Glucocorticoid and estrogen regulation of a rat T-kininogen gene.

We have examined the regulation of a rat T-kininogen gene by glucocorticoid and estrogen. Expression of the endogenous gene in a rat hepatoma cell line is increased 5-fold and 2-fold in response to dexamethasone and 17 beta-estradiol-3-benzoate, respectively. Various deletion constructs of the 5' region of an isolated T-kininogen gene were fused to a chloramphenicol acetyltransferase (CAT) gene and introduced into the hepatoma cells by electroporation. Analysis of the CAT activity in cell extracts after treatment with glucocorticoid or estrogen revealed that a fragment from -167 to +52 is sufficient to confer full induction. An additional deletion in this region was unresponsive, while a larger fragment (-612 to -100) linked to a heterologous promoter did result in regulated expression. These results suggested that the sequence responsible for the hormonal response was located at -167 to -100 from the transcription start site. This 67 bp region contains a consensus for the core sequence of the glucocorticoid responsive element (GRE) and the estrogen responsive element (ERE). Interestingly these elements are located within 7 bp of each other and both sequences overlap a 16 bp palindrome that may be important in hormone receptor-DNA recognition.

Animals↗