Biomedical subjects

H Ragg

Publications and source records attributed to H Ragg.

At least 19 recordsLinked to original sources

PubMed2001

Heparin cofactor II, antithrombin-beta and their complexes with thrombin in human tissues.

In the presence of glycosaminoglycans, thrombin is rapidly inactivated by two natural inhibitors secreted from liver: antithrombin (AT) is presumed to be the principal thrombin inhibitor in circulating blood, while for heparin cofactor II (HCII), a role outside circulation has been proposed. In this study, we show that HCII and AT differ with respect to their association with human tissues. Aside from brain, each of these inhibitors was found in sodium dodecyl sulphate (SDS) soluble extracts of various human organs, with a preponderance of HCII in placenta. AT levels, however, predominated in liver. Compared to plasma, the beta-variant of AT was found to be strongly enriched in human organs, while tissue-resident HCII did not differ in its electrophoretic mobility from the circulating form. In placenta, comparable amounts of HCII/thrombin and AT/thrombin complexes were detected, indicating that HCII may exert a thrombin regulating role in that organ under conditions of tissue or blood vessel damage. Transcripts coding for HCII and AT were detected in all tissues examined. The low levels of their mRNAs suggest that most of the tissue-associated thrombin inhibitor molecules originate from circulation and are retained in organs, possibly by specific receptors. The differential presence of HCII and AT in organs is in accordance with individual physiological roles of these inhibitors.

Antithrombin III↗

PubMed2001

Vertebrate serpins: construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses.

A combination of three independent biological features, genomic organization, diagnostic amino acid sites, and rare indels, was used to elucidate the phylogeny of the vertebrate serpin (serine protease inhibitor) superfamily. A strong correlation between serpin gene families displaying (1) a conserved exon-intron pattern and (2) family-specific combinations of amino acid residues at specific sites suggests that present-day vertebrates encompass six serpin gene families which evolved from primordial genes by massive intron insertion before or during early vertebrate radiation. Introns placed at homologous positions in the gene sequences in combination with diagnostic sequence characters may also constitute a reliable kinship indicator for other protein superfamilies.

Amino Acid Sequence↗

PubMed2001

Phylogenetic analyses of amino acid variation in the serpin proteins.

Phylogenetic analyses of 110 serpin protein sequences revealed clades consistent with independent phylogenetic analyses based on exon-intron structure and diagnostic amino acid sites. Trees were estimated by maximum likelihood, neighbor joining, and partial split decomposition using both the BLOSUM 62 and Jones-Taylor-Thornton substitution matrices. Neighbor-joining trees gave results closest to those based on independent analyses using genomic and chromosomal data. The maximum-likelihood trees derived using the quartet puzzling algorithm were very conservative, producing many small clades that separated groups of proteins that other results suggest were related. Independent analyses based on exon-intron structure suggested that a neighbor-joining tree was more accurate than maximum-likelihood trees obtained using the quartet puzzling algorithm.

Amino Acids↗

PubMed1999

Rapid changes in the exon/intron structure of a mammalian thrombin inhibitor gene.

The genomic organization of the heparin cofactor II (HCII) gene from rat and mouse was investigated and compared with their human counterpart. The genes share a common core structure consisting of five exons interrupted by four introns, but the mouse and rat gene reveal individual additional features. A unique differentially spliced exon is present in the 5'-untranslated region of the rat gene, which most probably has arisen de novo by point mutations in intronic sequences of the ancestor gene. In the mouse HCII gene, a novel intron/exon boundary has been created due to the presence of an additional DNA segment, which simultaneously provides a 3'-splice site and a polypyrimidine stretch leading to an alternatively used exon of increased size. Our data suggest that, in contrast to most other mammalian genes, the exon/intron pattern of the gene coding for HCII is in dynamic evolution.

Alternative Splicing↗

PubMed1994

Secondary thrombin-binding site, glycosaminoglycan binding domain and reactive center region of leuserpin-2 are strongly conserved in mammalian species.

Using a combination of conventional and inverse polymerase chain reaction the cDNA structure coding for the thrombin inhibitor leuserpin-2 from rat has been determined. The rat genome specifies leuserpin-2 mRNAs differing in their 5'-untranslated regions probably due to alternative splicing of the primary transcript. Comparisons of partial sequences from five different mammalian species show that the secondary thrombin binding site, the glycosaminoglycan binding domain and the reactive center region of the inhibitor are strongly conserved.

Amino Acid Sequence↗

PubMed1990

Glycosaminoglycan-mediated leuserpin-2/thrombin interaction. Structure-function relationships.

Two recently identified structural elements important for glycosaminoglycan-mediated activation of human leuserpin-2 (hLS2) were investigated in detail by functional analysis of variants secreted by transiently transfected COS cells. Highly specific requirements with respect to the nature of the involved amino acids as well as to their spatial arrangements were found to be crucial for efficient activation of hLS2 by dermatan sulfate. In contrast, binding and activation of hLS2 by heparin seem to be determined mainly by the positive charge density of the involved inhibitor segment. A dimeric repeat enriched in acidic amino acids turned out to exert a dual role with respect to structure and function of hLS2. First, in the absence of functional activators the negatively charged dimer interacts intramolecularly with the glycosaminoglycan-binding site. Second, the acidic dimer is instrumental in glycosaminoglycan-mediated activation of hLS2. The monomers constituting the acidic dimer are functionally not equivalent.

Amino Acid Sequence↗

PubMed1990

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells.

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.

Amino Acid Sequence↗

PubMed1990

On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans.

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.

Amino Acid Sequence↗

PubMed1988

Structure and expression of the gene coding for the human serpin hLS2.

We have analyzed genomic clones encoding human leuserpin 2 (hLS2). The gene covers about 14.5 kilobases and consists of 5 exons and 4 introns. The genes coding for hLS2, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and rat angiotensinogen share an equivalent exon-intron structure and therefore constitute a distinct subgroup within the serpin gene family, which otherwise displays a highly variable exon-intron pattern. With the exception of a segment in the second exon, the sequence similarity of the genes coding for hLS2 and alpha 1-antitrypsin extends to all exons including one encoding the 5'-untranslated sequences. The implications of these findings with respect to the genesis of the amino-terminal heterogeneity in the serpin family are discussed.

Amino Acid Sequence↗

PubMed1987

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.

Antithrombin III↗

PubMed1986

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.

Amino Acid Sequence↗

PubMed1985

A 46-nucleotide promoter segment from an IFN-alpha gene renders an unrelated promoter inducible by virus.

A hybrid gene consisting of the human IFN-alpha 1 promoter and a beta-globin transcription unit is expressed correctly only after viral induction. To determine the region required for inducibility, 25 hybrid promoters consisting of varying upstream IFN-alpha 1 and downstream beta-globin promoter moieties were analyzed, and 5'-deletion analysis was performed on an inducible hybrid promoter. An IFN promoter region from position -109 to -64 conferred maximal inducibility on downstream beta-globin promoter segments and even on the intact beta-globin promoter. This region is strikingly conserved among human IFN-alpha and -beta genes. As constitutive expression of the beta-globin gene was not diminished by placing IFN promoter fragments in various positions, induction is attributed largely to positive, rather than to negative control.

Animals↗

PubMed1983

UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselinum hortense.

DNAs complementary to poly(A)(+) mRNAs from UV-irradiated cell suspension cultures of parsley (Petroselinum hortense) were inserted into pBR322 and used to transform Escherichia coli strain RR1. A clone containing a DNA complementary to chalcone synthase mRNA was identified by hybrid-selected and hybrid-arrested translation. Large and rapid changes in the amount of chalcone synthase mRNA in response to irradiation of the cells was detected by RNA blot hybridization experiments. The pattern of changes coincided with that previously determined for the rate of chalcone synthase synthesis as measured either in vivo or with polyribosomal mRNA in vitro. The results are consistent with the hypothesis that induction of chalcone synthase by UV light is due to a transient increase in the rate of synthesis of chalcone synthase mRNA.

Journal Article↗

PubMed1982

Structure and expression of human IFN-alpha genes.

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-alpha genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-alpha 1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-beta and IFN-gamma are glycosylated. Because E. coli cells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1 and could be propagated for at least several months.

Base Sequence↗

PubMed1982

Structure and expression of human alpha-interferon genes.

cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.

Amino Acid Sequence↗

PubMed1982

Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana.

Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

Acyltransferases↗

PubMed1981

Coordinated regulation of 4-coumarate:CoA ligase and phenylalanine ammonia-lyase mRNAs in cultured plant cells.

4-Coumarate:CoA ligase (EC 6.2.1.12) from cell suspension cultures of parsley (Petroselinum hortense) was extensively purified. The enzyme behaved as a monomer with a molecular weight of approximately 60,000. A rabbit antiserum to the purified enzyme was obtained and used to determine the rates of 4-coumarate:CoA ligase synthesis under various conditions of induction, such as short term or continuous irradiation and treatment of the cells with an "elicitor" preparation from a fungal pathogen. In all cases, the time course of changes in the rate of synthesis, measured both in vivo and in vitro, was very similar for 4-coumarate:CoA ligase and a closely related enzyme, phenylalanine ammonia-lyase (EC 4.3.1.5). The results suggest that the mRNA activities encoding the two enzymes are regulated in a coordinated manner.

Ammonia-Lyases↗

PubMed1980

Messenger RNA coding for phenylalanine ammonia-lyase. Characterization and partial purification from cell suspension cultures of Petroselinum hortense.

The mRNA coding for phenylalanine ammonia-lyase was partially purified from irradiated cell suspension cultures of parsley (Petroselinum hortense). The product of cell-free translation of the mRNA in a reticulocyte lysate was isolated by immunoprecipitation and compared with the native enzyme subunit. Evidence for the identity, or at least a great similarity, of both was provided by tryptic-peptide and gel-electrophoretic analyses. Under partially denaturing conditions, phenylalanine ammonia-lyase mRNA sedimented as a 20--21-S molecule in a sucrose gradient and had an apparent molecular weight of about 1.05 x 10(6) on a polyacrylamide gel. Approximately two-thirds of the polynucleotide sequence of the mRNA were estimated to be required as coding sequence for the enzyme. We suggest that phenylalanine ammonia-lyase mRNA is unlikely to code for more than of three coordinately induced enzymes.

Ammonia-Lyases↗