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Biomedical subjects

H Will

Publications and source records attributed to H Will.

At least 145 records · Page 8Linked to original sources
PubMed1991

Effects of monoclonal antibodies on tissue-type plasminogen activator (t-PA) binding to lysine, fibrin and heparin and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity.

The effects of 4 monoclonal antibodies against human tissue-type plasminogen activator (t-PA) on binding of t-PA to lysine, fibrin, and heparin, and on fibrin-mediated activation of one-chain t-PA-amidolytic activity were investigated. The association constants of the antibodies were determined in a direct assay to be equal to 0.125 l/nmol, 0.225 l/nmol, 0.4 l/nmol, and 0.5 l/nmol for mAB 5, mAB 16, mAB 25, and mAB 31, respectively. All 4 monoclonal antibodies inhibited binding of intact t-PA to lysine-Sepharose and fibrin, and they suppressed fibrin-mediated activation of one-chain t-PA-amidolytic activity. Binding analysis demonstrated that mAB 25 inhibited t-PA binding to lysine-Sepharose and to fibrin as well as fibrin-mediated enhancement of one-chain t-PA-amidolytic activity in a competitive manner with inhibitor constants of 5 nmol/l, 3 nmol/l and 10 nmol/l, respectively. It was also shown that free lysine counteracts the association of t-PA with the antibodies. Binding of t-PA to heparin is only moderately affected by the 4 antibodies. Since t-PA possesses two homologous kringle domains which contain fibrin (lysine) binding sites, the results underline the importance of a lysine binding site for fibrin binding by intact t-PA and show that the binding of the enzyme to fibrin and lysine is mediated by the same binding site of a kringle domain. The parallel effects of antibodies on fibrin binding and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity proves that the site of fibrin binding is identical with the site of fibrin activation. The binding site of heparin apparently differs from lysine and fibrin binding sites.

Antibodies, Monoclonal↗
PubMed1991

Hepatitis B virus and hepatocellular carcinoma: a possible role for the viral transactivators.

Several epidemiological studies have demonstrated a link between chronic B virus infection and primary hepatocellular carcinoma (PHC). HBV DNA sequence integrations into the host cell genome have often been observed in hepatocarcinoma tissues. However, since only in a few cases of PHC the target of HBV-DNA insertion has been identified, alternative mechanisms for HBV-induced hepatocyte transformation have been investigated. Like many other DNA viruses, the hepatitis B virus bears a transactivational potential. Both full length and truncated versions of HBV X protein are able to influence the expression of cellular nuclear protooncogenes c-fos and c-myc. A second transcriptional activator is encoded by the PreS/S region of HBV, but its activity on viral and cellular genes become evident only after dislocations from its downstream sequences. Thus, HBV is able to influence infected cell growth and differentiation using both native proteins, newly generated truncated proteins and virus-cell fusion polypeptides.

Carcinoma, Hepatocellular↗
PubMed1991

Effect of N-linked glycosylation on hepatic lipase activity.

Hepatic lipase (HL) is a secretory protein synthesized in hepatocytes and bound to liver endothelium. Previous studies have suggested that HL N-linked glycans are required for catalytic activity. To directly test this hypothesis, Xenopus laevis oocytes were used to express native rat HL or HL lacking one or both N-linked glycosylation sites. The expressed and secreted native HL had an apparent molecular mass of 53 kDa, consistent with purified rat liver HL. The mutant lacking both glycosylation sites, while poorly secreted, had an apparent molecular mass of 48 kDa, the same size observed for HL after enzymatic removal of N-linked oligosaccharides. Mutants lacking one of the two sites were intermediate in size and showed reduced secretion. Each of these expressed and secreted proteins had full catalytic activity that was inhibited by antisera to rat HL. Thus, N-linked glycosylation of rat HL, while important to lipase secretion, is not essential for the expression of lipase activity.

Animals↗
PubMed1990

Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination.

Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains. When these live recombinant bacteria were administered i.p. to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies. Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used. With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization. The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p. and oral immunization is consistent with the induction of functional T cell help.

Administration, Oral↗
PubMed1990

Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.

Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.

Amino Acid Sequence↗
PubMed1990

Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator.

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.

Amino Acid Sequence↗
PubMed1990

Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies.

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.

Antibodies, Monoclonal↗
PubMed1990

Isolation and characterization of the human hepatic lipase gene.

Overlapping bacterial phage and cosmid genomic clones were isolated spanning an area of approximately 60 kilobases that contains the human hepatic lipase (HL) gene. It is composed of 9 exons spanning approximately 35 kilobases of DNA. The entire coding regions, the 5'-flanking sequences, and the exon-intron junctions were sequenced. The intron positions correspond to those of human lipoprotein lipase and canine pancreatic lipase, supporting the concept that these genes constitute a dispersed gene family of lipases and have evolved by duplication of a common ancestral gene. A region of the HL gene, which displays a significant homology with various other lipolytic enzymes and contains the putative catalytic site serine residue of HL, was encoded by exon 4. A major transcription start site of the human HL gene was located by primer extension analysis, 43 nucleotides upstream of the translation initiation codon. Two possible promoter elements were located 25 and 63 nucleotides upstream of the transcription initiation site: a "TATA" box-like sequence, TAATA, and a sequence found in the promoter region of many liver-specific genes, AGGTTAATTATTAAT. In addition, sequences homologous to glucocorticoid and cAMP-responsive elements were identified in the 5'-nontranscribed region.

Amino Acid Sequence↗
PubMed1990

Hepatic lipase: site-directed mutagenesis of a serine residue important for catalytic activity.

Hepatic lipase (HL) is a member of the lipoprotein lipase/pancreatic lipase gene family and is believed to function in processing of intermediate and high density lipoproteins. As a lipase, HL is presumed to have a lipid interfacial binding domain, distinct from the esterase catalytic site, orienting the enzyme at aqueous-lipid interfaces and resulting in activation of esterase activity. However, the structural domains responsible for these separate functions have not been identified. Amino acid sequence homology to serine proteases, thioesterases and other lipases, identified Ser147 of rat HL as part of a highly conserved element in an esterase gene family. In order to better define the function of this domain in HL, site-directed mutagenesis was utilized to produce mutant cDNAs with amino acid substitutions for Ser147, Ser133, or Ser228. Following injection of Xenopus oocytes with SP6 transcripts for normal or mutant HL, media from the oocytes were assayed for lipolytic activity and immunoprecipitable HL protein. Mutations of Ser133 and Ser228 produced no decrease in activity whereas the mutant protein in which Ser147 was replaced with glycine had little, if any activity against emulsified triolein substrates. Replacing HL Ser147 with glycine also resulted in a protein with little or no measurable activity for tributyrin, a substrate which does not provide a lipid interface. These results suggest that Ser147 in rat HL is either located at the catalytic site or is required for maintaining the structural integrity of the catalytic site.

Animals↗
PubMed1990

Human anti-p68 autoantibodies recognize a common epitope of U1 RNA containing small nuclear ribonucleoprotein and influenza B virus.

Autoantibodies from patients with systemic rheumatic diseases were used to map antigenic sites on the 68-kD autoantigen (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein (snRNP) particles. With truncated recombinant fusion proteins and synthetic peptides, a subset of anti-p68 autoantibodies was found to recognize the amino acid sequence motif Glu-Arg-Lys-Arg-Arg (ERKRR). To investigate the possible involvement of epitopes shared by microbial antigens and host self-components in initiation of autoimmunity (molecular mimicry), a sequence data bank was screened for proteins containing an amino acid motif identical or related to ERKRR. The identical motif was found on the M1 matrix protein of influenza B viruses, and affinity-purified human anti-ERKRR autoantibodies recognized this epitope also in the viral amino acid sequence context. The common epitope recognized by human autoantibodies suggests that influenza B virus infection may play a role in initiation of the anti-p68 and anti-(U1)RNP autoimmune response.

Animals↗
PubMed1990

Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection.

All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.

Cell Fractionation↗
PubMed1990

A new hepatitis B virus variant in a chronic carrier with multiple episodes of viral reactivation and acute hepatitis.

To examine whether there are HBV variants not yet described which cannot express HBe due to a mutation in the pre-C region, and, if they exist, whether they cause a particular course of infection and disease, we analyzed the HBV genomes of a HBs/anti-HBe positive chronic carrier who had several episodes of acute reexacerbations of chronic hepatitis with at least two viremic phases. Direct sequencing of the precore/core and the pre-S regions of the HBV sequences of both viremic phases amplified by the polymerase chain reaction revealed that they were very similar to each other but substantially divergent from published HBV genomes. Both virus populations contained a mutation in the first nucleotide of the pre-C translation initiation codon (AUG/CUG) which prevents HBeAg expression. These data demonstrate the existence of a new HBV variant which can enter a latent phase from which it can be reactivated with acute reexacerbation of liver inflammation.

Adult↗
PubMed1990

A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B.

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

Adult↗
PubMed1990

Latency and reactivation of a precore mutant hepatitis B virus in a chronically infected patient.

In human patients infected with hepatitis B virus (HBV) seroconversion from HBe to anti-HBe often signals virus elimination. Occasionally, a second viremic phase is observed which may be due to superinfection with a variant or reactivation of a latent virus. To differentiate between these two possibilities we investigated the nucleotide sequences of virus populations from sera of a chronically infected patient who had two distinct viremic phases, one e-antigen positive and one anti-HBe antibody positive. By direct sequencing of amplified HBV C- and pre-S gene sequences we found that the viruses in the two populations differed by only two point mutations, one of which prevents expression of precore derived e-antigen. In the nonviremic phase viral DNA and antigen were found in the liver but nucleocapsid protein expression appeared drastically downregulated. These data demonstrate that HBV can enter a latent phase with low expression of core protein and selection for HBV variants which cannot synthesize e-antigen. This suggests that e-antigen expression can be a critical target for virus elimination and that loss of its expression can prolong chronicity and provoke latency of HBV infection.

Antigens, Viral↗
PubMed1990

Recognition of a hepatitis B virus nucleocapsid T-cell epitope expressed as a fusion protein with the subunit B of Escherichia coli heat labile enterotoxin in attenuated salmonellae.

Two overlapping T-cell sites of the nucleocapsid antigen (HBc) of Hepatitis B Virus (HBV) (amino acids (aa) 120-140) and a B-cell epitope of the pre-S(2) region of the HBV surface antigen (aa 133-140) were expressed as a fusion protein with the subunit B of Escherichia coli heat labile enterotoxin (LT-B) in attenuated salmonellae (aroA Salmonella dublin SL1438). When Balb/c (haplotype H-2d) mice were fed salmonellae expressing LT-B or the LT-B/HBV fusion protein they developed serum IgG anti-LT-B antibodies and splenic cells reactive to LT-B. C57BL/10 (H-2b), in contrast, showed anti-LT-B antibody titres, but no splenic cell priming to LT-B. Neither in Balb/c nor in C57BL/10 mice could an antibody response to the fused HBV antibody binding site be demonstrated. In C57BL/10, however, an HBc T-cell epitope fused to LT-B primed a splenic cell response to an analogous synthetic peptide (HBc aa 121-145) in four out of five mice after three oral immunizations. This is the first description of the priming of a cellular immune response to a defined heterologous epitope expressed in attenuated salmonellae and delivered by the oral route.

Animals↗
PubMed1990

Major autoantigenic sites of the (U1) small nuclear ribonucleoprotein-specific 68-kDa protein.

A 68-kDa protein associated with (U1)snRNP is a major target for human autoantibodies to small ribonucleoprotein particles (snRNP) prevalent in a variety of inflammatory rheumatic diseases. The epitopes recognized by these antibodies were mapped by expression of subfragments of p68 cDNA in Escherichia coli and testing of the corresponding recombinant proteins for immunoreactivity with sera of patients with autoimmune diseases. Three of four antigenic regions were analysed in detail. The immunodominant autoantigenic region was found to coincide with the RNA-binding domain of the p68 protein and was shown to contain a nested set of overlapping discontinuous epitopes. Two additional non-overlapping major antigenic domains were localized in the carboxy-terminal half of the p68 protein. Each of these two carboxy-terminal domains was shown to contain more than one conformation-dependent epitope. Taking into account previous mapping studies, the data demonstrate that p68 contains at least four antigenic regions, each of which harbours multiple epitopes which are recognized in a patient-specific manner.

Amino Acid Sequence↗