Biomedical subjects

A Wilmen

Publications and source records attributed to A Wilmen.

10 recordsLinked to original sources

PubMed2005

In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor.

BAY 59-7939 is an oral, direct Factor Xa (FXa) inhibitor in development for the prevention and treatment of arterial and venous thrombosis. BAY 59-7939 competitively inhibits human FXa (K(i) 0.4 nm) with > 10 000-fold greater selectivity than for other serine proteases; it also inhibited prothrombinase activity (IC(50) 2.1 nm). BAY 59-7939 inhibited endogenous FXa more potently in human and rabbit plasma (IC(50) 21 nm) than rat plasma (IC(50) 290 nm). It demonstrated anticoagulant effects in human plasma, doubling prothrombin time (PT) and activated partial thromboplastin time at 0.23 and 0.69 microm, respectively. In vivo, BAY 59-7939 reduced venous thrombosis (fibrin-rich, platelet-poor thrombi) dose dependently (ED(50) 0.1 mg kg(-1) i.v.) in a rat venous stasis model. BAY 59-7939 reduced arterial (fibrin- and platelet-rich) thrombus formation in an arteriovenous (AV) shunt in rats (ED(50) 5.0 mg kg(-1) p.o.) and rabbits (ED(50) 0.6 mg kg(-1) p.o.). Slight inhibition of FXa (32% at ED(50)) reduced thrombus formation in the venous model; to affect arterial thrombosis in the rat and rabbit, stronger inhibition of FXa (74%, 92% at ED(50)) was required. Calculated plasma levels in rabbits at the ED(50) were 14-fold lower than in the rat AV shunt model, correlating with the 14-fold lower IC(50) of FXa inhibition in rabbit compared with rat plasma; this may suggest a correlation between FXa inhibition and antithrombotic activity. Bleeding times in rats and rabbits were not significantly affected at antithrombotic doses (3 mg kg(-1) p.o., AV shunt). Based on these results, BAY 59-7939 was selected for clinical development.

Animals↗

PubMed2001

Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis.

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.

Animals↗

PubMed2000

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal nontransformed tissues is unknown. We report here that chronic blockade of TRAIL in mice exacerbated autoimmune arthritis, and that intraarticular TRAIL gene transfer ameliorated the disease. In vivo, TRAIL blockade led to profound hyperproliferation of synovial cells and arthritogenic lymphocytes and heightened the production of cytokines and autoantibodies. In vitro, TRAIL inhibited DNA synthesis and prevented cell cycle progression of lymphocytes. Interestingly, TRAIL had no effect on apoptosis of inflammatory cells either in vivo or in vitro. Thus, unlike other members of the tumor necrosis factor superfamily, TRAIL is a prototype inhibitor protein that inhibits autoimmune inflammation by blocking cell cycle progression.

Animals↗

PubMed1998

The genomic organization of the human GLP-1 receptor gene.

The genomic organization of the human gene encoding the receptor for glucagon-like peptide-1 (GLP-1 (7-37)/(7-36) amide) was analyzed to reveal the relationship to other G-protein-coupled receptors. The coding sequence of the GLP-1 receptor is interrupted by 12 introns. These introns are uniformly distributed within the open reading frame. The length of the introns varies between 6.6 kb and 100 bp, in contrast to the relative constant length of 100 bp of the exons. All of the exon/intron splice junctions characterized followed the consensus GT-AG rule. A comparison of the genomic structure with other related receptor genes indicates that the exon/intron organization is well-conserved among the VIP/ glucagon/secretin receptor family.

3' Untranslated Regions↗

PubMed1997

Five out of six tryptophan residues in the N-terminal extracellular domain of the rat GLP-1 receptor are essential for its ability to bind GLP-1.

Oligonucleotide-directed mutagenesis was utilized to investigate the requirement of tryptophan residues located in the N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor for the ability to bind its ligand and to induce cAMP generation. W39, W72, W87, W91, W110, and W120 were mutated into alanine. Two of the six tryptophan residues, W72 and W110, are highly conserved within the receptor subfamily. After transfection of mutated cDNAs in COS-7 or CHL cells, it appeared that mutant W87 A bound [125I] GLP-1 with the same affinity as wild-type receptor and induced signal transduction to a comparable extent. In contrast, mutant receptors W39A, W72A, W91A, W110A, and W120A lost the ability to bind [125I] GLP-1. Because all mutated receptor cDNAs were transcribed on RNA level (Northern blot) and the receptor proteins were expressed at the plasma membrane level (Western blot), it is concluded that with the exception of W87 all trytophan residues are essential for receptor ligand interaction. This indicates the significance of hydrophobic interactions within the N-terminal domain of the GLP-1 receptor.

Amino Acid Sequence↗

PubMed1996

The isolated N-terminal extracellular domain of the glucagon-like peptide-1 (GLP)-1 receptor has intrinsic binding activity.

The glucagon-like peptide 1 (7-37)/(7-36) amide (GLP-1) receptor belongs to a new subclass of seven transmembrane domain, G-protein coupled receptors comprising several receptors for peptide hormones. The receptors of this family share many common motifs including a relatively large N-terminal extracellular domain. The GLP-1 receptor is presently attracting much attention, since it is the target protein of the antidiabetic gut hormone GLP-1. To establish the functional significance of the N-terminal part of the GLP-1 receptor for ligand binding, the extracellular domain was isolated and purified. Utilizing CHL cells expressing the cloned GLP-1 receptor, we demonstrate that the isolated, solubilized N-terminal part of the receptor protein competes for GLP-1 binding with the intact wild-type receptor. Moreover, in cross-linking experiments radiolabeled GLP-1 was covalently attached to the isolated N-terminus, thereby demonstrating direct physical interaction of both components. By Western blot analysis two specific bands were detectable, representing the N-terminal receptor protein in the presence or absence of bound ligand. These data underline the significance of the N-terminal domain of the GLP-1 receptor for ligand binding.

Amino Acid Sequence↗

PubMed1996

The chromatin of the Saccharomyces cerevisiae centromere shows cell-type specific changes.

We have analysed the centromeric chromatin from chromosome XIV of Saccharomyces cerevisiae at different stages of mitosis with the help of mutants of the cell division cycle. The pattern of centromeric chromatin in cells arrested using cdc20-1, tub2-401 and cdc15-1 alleles was indistinguishable from that of vegetatively growing cells, indicating that the centromeric complex is constitutively present during mitosis and possibly throughout the entire cell cycle. In contrast chromatin isolated from G0 cells and spores exhibited distinct differences in centromeric chromatin probably due to structural rearrangements of the centromeric complex. In particular the alterations found in spores are indicative of an inactive centromeric complex. The differences in centromeric chromatin in spores do not reflect a general reorganisation of the chromatin in this cell type, as the chromatin structure of the PHO3/PHO5 locus in spores was found to be identical to that in vegetative cells under repressed conditions. Thus the structural analysis of the centromere in different cell types provides evidence about the requirement of CEN DNA/protein complexes in different cell types and in different stages of the cell cycle.

Acid Phosphatase↗

PubMed1996

Exchange of W39 by A within the N-terminal extracellular domain of the GLP-1 receptor results in a loss of receptor function.

The proglucagon-derived glucagon-like peptide-1 (GLP-1) secreted by the L-cells exerts an insulinotropic effect at pancreatic beta-cells. The GLP-1 receptor belongs to a new subfamily of the superfamily of seven transmembrane, G-protein-coupled receptors (7 TM receptors). We show that a single point mutation within a nonconserved motif of the N-terminal, extracellular domain of the GLP-1 receptor results in a dramatic impairment of receptor function. Thus, substitution of W39 by A or F is followed by a loss of GLP-1 binding. Exchange of K38 with A (mutant K) slightly decreased GLP-1 binding affinity. Replacement of the negatively charged Q37 by K and K38 by A, which is identical with a shift of the positively charged K one position upstream, resulted in a receptor mutant able to bind GLP-1 with higher affinity as the wild-type receptor and mutant K. Therefore, the presence of an imidazol ring structure in the investigated receptor region is necessary for an intact receptor function Furthermore, a positive charge at this location is important for the receptor-ligand interaction.

Amino Acid Sequence↗

PubMed1994

The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.

The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes.

Base Sequence↗

PubMed1993

Cpf1 protein induced bending of yeast centromere DNA element I.

The centromere complex is a multicomponent structure essential for faithful chromosome transmission. Here we show that the S. cerevisiae centromere protein Cpf1 bends centromere DNA element I (CDEI) with the bend angle ranging from 66 degrees to 71 degrees. CDEI DNA sequences that carry point mutations which lead to reduced Cpf1 binding affinity and in vivo centromere activity are still able to show bending. The Cpf1 induced bend is directed towards the major groove with the bend centre located in CDEI. An intrinsic bend cannot replace the Cpf1 induced DNA bend for in vivo centromere function. An in vivo phasing experiment suggests that both the distance and the correct spatial arrangement of the CDEI/Cpf1 complex to CDEII and CDEIII are important for optimal centromere function.

Base Sequence↗