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Biomedical subjects

H Will

Publications and source records attributed to H Will.

At least 127 records · Page 7Linked to original sources
PubMed1991

Characterization of a tissue-type plasminogen activator from porcine urine.

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an Mr of 65,000 similar to the Mr of human t-PA (64-70,000) but distinct from the Mr of human u-PA (55,000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.

Amiloride↗
PubMed1991

Characterization of cDNA encoding the mouse hepatic triglyceride lipase and expression by in vitro translation.

A cDNA coding for the mouse hepatic triglyceride lipase has been isolated from a mouse liver cDNA library. The nucleotide sequence of the cDNA shows an open reading frame encoding a polypeptide of 510 amino acids that is 91.5% and 86% homologous to rat and human hepatic lipase, respectively. The most drastic protein sequence divergence is found at the carboxyterminal end which was speculated to harbour one heparin-binding site. By in vitro translation of cRNA in the presence of pancreatic membranes the hepatic lipase was shown to be glycosylated and to have an electrophoretic mobility of 53 kDa.

Amino Acid Sequence↗
PubMed1991

Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cmyc protooncogene at the transcriptional level.

The products of the human hepatitis B virus (HBV) and woodchuck hepatitis B virus X genes (pXs) transactivate homologous and heterologous genes including the HBV-X and core promoters, the human immunodeficiency viruses 1 (HIV-1) and 2 (HIV-2) long terminal repeats and the beta interferon regulatory sequences. We report here that pX is also able to influence the expression of both extrachromosomal transfected c-myc regulatory sequences and endogenous c-myc gene. pX acts by increasing transcription of the c-myc gene and do not affect c-myc mRNAs stability. The presence of the first AUG of the X-ORFs is indeed necessary for the production of an active pX. The very carboxyterminus of the pX protein is dispensable for this transactivating activity and at least one domain important for its action is located between aminoacids 103 and 117.

Adenoviruses, Human↗
PubMed1991

Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.

Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus.

Adolescent↗
PubMed1991

Synthesis in Vibrio cholerae and secretion of hepatitis B virus antigens fused to Escherichia coli heat-labile enterotoxin subunit B.

A simple and effective electroporation method for the transformation of Vibrio cholerae with nonmobilizable plasmids is described. Expression plasmids directing the synthesis of fusion proteins with the subunit B of Escherichia coli heat-labile enterotoxin B (LT-B) were transformed into nontoxinogenic V. cholerae vaccine strains. A protein consisting of two overlapping immunodominant antibody-binding sites of the hepatitis B virus (HBV) middle surface antigen fused to the C terminus of full-length LT-B was secreted into the supernatant of V. cholerae cultures, whereas two other LT-B/HBV fusion proteins were mostly retained within the cells or rapidly degraded in the culture supernatant. While the secretion of fusion proteins with cholera toxin subunit B (CT-B) from V. cholerae has been described, this is to our knowledge the first report describing extracellular secretion of defined foreign epitopes fused to LT-B in V. cholerae. The fusion of guest epitopes to LT-B or CT-B and secretion in V. cholerae could be an interesting system to rapidly produce pure fusion proteins for immunisation, functional studies or diagnostic procedures. An LT-B/pre-S2 fusion protein purified from the supernatant of recombinant V. cholerae induced serum IgG antibodies against LT-B and against the HBV middle surface antigen in mice after parenteral and oral immunisation.

Animals↗
PubMed1991

Acetylcholine receptor-specific T-lymphocyte clones in the normal human immune repertoire: target epitopes, HLA restriction, and membrane phenotypes.

Potentially autoimmune T-lymphocyte lines specific for the nicotinic acetylcholine receptor of the neuromuscular junction have been isolated previously from patients with myasthenia gravis. We report on the isolation and expansion of T cells specific for the acetylcholine receptor of Torpedo californica or for a recombinant mammalian acetylcholine receptor alpha chain peptide (X4), from the peripheral blood of 11 healthy donors. Two major T-cell epitopes, located between amino acid positions 44-104 and 141-172, were identified using a panel of overlapping mammalian alpha chain fusion proteins. Most T lines recognized the acetylcholine receptor epitopes in the molecular context of HLA-DR molecules. Unexpectedly, all the T. californica acetylcholine receptor-specific T lines obtained from one DR4 (DRw53), DQw3 donor and two DR4, w8 (DRw53), DQw3 donors were restricted by DRw53 product(s). Using DR gene-transfected L cells as antigen presenters, in 4 lines, a close relationship between the recognized epitope and the restricting DR element was revealed. The membrane phenotype of the T. californica acetylcholine receptor-and X4-specific T lines was predominantly CD4+CD8-, with some CD4+CD8+ components. It did not significantly differ from that of control, tuberculin purified protein derivate-specific T lines raised from the same donors. These findings are in harmony with previous ones demonstrating the presence of potentially autoimmune T-lymphocyte clones within normal immune repertoires.

Antigens, CD↗
PubMed1991

Mechanism, kinetics, and role of duck hepatitis B virus e-antigen expression in vivo.

No duck hepatitis B virus (DHBV) pre-C transcript has been identified so far, and neither the interrelationship of e-antigen (DHBeAg) with the expression of other viral antigens or virus replication nor its function is known. In this study we identified in infected livers a minor transcript from which the precursor protein of DHBeAg could be synthesized. Mutation of the first AUG on this transcript abolished expression of DHBeAg. DHBV genomes containing this mutation were infectious in Pekin ducks, the kinetics of pre-S envelope protein expression and virus secretion were not significantly different from wild-type, and the mutant genomes did not revert to wild-type to a detectable level after several passages. In contrast to pre-S protein, the level of DHBeAg in the serum was independent of the level of viremia, accumulated gradually to a high and constant level after a lag phase, and was also easily detectable in a mixed infection containing less than 0.1% of wild-type in a pre-C mutant virus containing inoculum. These data indicate that precore protein is synthesized from a minor pre-C mRNA with translation initiation at the pre-C AUG codon, and leads to high levels of DHBeAg rather late in infection. High levels of DHBeAg can even be produced efficiently by a very small subpopulation of wild-type virus in a mixed infection with predominantly pre-C mutant virus. Lack of DHBeAg appears to have no effect on DHBV viability and kinetics of virus secretion into the bloodstream when ducklings are infected with the pre-C AUG mutant virus a few days after birth.

Animals↗
PubMed1991

Characterization of infectious and defective cloned avian hepadnavirus genomes.

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.

Amino Acid Sequence↗
PubMed1991

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area.

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.

Adult↗
PubMed1991

High prevalence and heterogeneity of HBV preC mutants in anti-HBe-positive carriers with chronic liver disease in southern Italy.

In this study we have investigated the prevalence and type of preC mutants in anti-HBe/HBV-DNA-positive patients with chronic hepatitis B in an endemic area. HBV-DNA from sera of 42 anti-HBe chronic HBV carriers was amplified by PCR and the preC region was directly sequenced. With one exception, all patients tested were found to be infected with viruses containing mutations in the preC region that predictably prevent precore and e antigen expression. Thirty-one patients were infected with HBV containing a stop codon; two had a mixture of wild-type and preC stop codon mutant; three had preC mutants with mutations in the translation initiation codon and two of them an additional stop codon; four had a frameshift mutation, and one had two stop codons. One patient was infected with a virus showing a mutation creating only an amino acid exchange which could not prevent HBeAg expression. The data obtained indicate a higher prevalence and heterogeneity of preC mutants in our geographical area than recognized so far in anti-HBe-positive carriers with chronic hepatitis B.

Base Sequence↗
PubMed1991

Replicating and virion secreting hepatitis B mutant virus unable to produce preS2 protein.

Mutant HBV genomes unable to produce preS2 protein were identified in the serum of a highly viremic chronic carrier. By direct sequencing of the amplified preS region and by sequencing of 50 cloned amplified preS DNA fragments all molecules were found to have deleted the preS2 translation initiation codon and three amino acids 54 nucleotides downstream thereof. In addition, numerous amino acid changes, predominantly located between both deletions, were revealed. HBV-DNA genomes containing the mutated preS sequences were shown to be replication competent and to secrete efficiently virions that were morphologically and by the type of DNA encapsidated not distinguishable from wild-type virus. These data demonstrate that HBV with mutated preS sequences and unable to express preS2 protein can occur as a dominant or exclusive virus population in a highly viremic chronic carrier. The data also show that expression of the preS2 protein is not essential for HBV replication, virion morphogenesis and secretion.

Adult↗
PubMed1991

Selection for a pre-C stop codon mutation in a hepatitis B virus variant with a pre-C initiation codon mutation during interferon treatment.

Hepatitis B virus (HBV) variants with a stop codon, a mutated translation initiation codon or other mutations in the pre-C region which prevent e-antigen expression are highly prevalent in anti-HBe chronic carriers and can be positively selected from a mixed virus infection. Our laboratories recently described pre-C variants with two pre-C mutations which prevent HBeAg expression. Here we have investigated whether there is a selective pressure for acquisition of the second pre-C mutation. By direct sequencing of amplified HBV DNA from sera of a chronic carrier taken during a 6-year follow-up, we found that genomes of a virus population virtually all had a pre-C translation initiation codon mutation and about 50% had an additional stop codon mutation. With the onset of interferon treatment, the genomes with the stop codon mutation increased to more than 95% while the frequency of the translation initiation codon mutation in all genomes remained constant. These data indicate positive selection (possibly immune-mediated and HBeAg-targeted) for a second pre-C mutation. This putative enhancement of negative translational control may be present because a pre-C translation initiation codon mutation cannot totally prevent HBeAg expression and is therefore less frequent.

Adult↗
PubMed1991

Repetitive P68-autoantigen specific epitopes recognized by human anti-(U1) small nuclear ribonucleoprotein autoantibodies.

The major target of anti-(U1)snRNP autoantibodies, a serological marker of patients with mixed connective tissue disease and related rheumatic disorders, is a 68 kDa protein (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein particles. With recombinant p68 fusion proteins, multiple autoepitopes have been identified, and one of these has been mapped to the pentamer sequence ERKRR, which is located within antigenic domain A in the amino-terminal half of p68. The lysine residue (K) of this epitope can be replaced by isoleucine without loss of autoantibody binding. Here we have investigated whether other variants of this epitope are present on the p68 autoantigen and if these are recognized by anti-p68 autoantibodies. We identified four related motifs in the carboxy-terminal half of the p68-protein, and three of these (all containing glutamic acid instead of lysine (ERERR] mapped to the previously characterized autoantigenic domains C and D. Immunoreaction of anti-ERKRR autoantibodies, affinity-purified from domain A with recombinant fusion proteins containing either domain C or domain D of p68, revealed that anti-ERKRR autoantibodies cross-react with the ERERR-motifs. This finding, which was confirmed by competitive inhibition-ELISA with solid-phase coupled domain A-, C- and D-fusion proteins and ERKRR-containing synthetic peptides as competitors, suggests that a subset of patient autoantibodies is directed against repetitive structures on a single snRNP component.

Amino Acid Sequence↗
PubMed1991

Regulatory sequences of duck hepatitis B virus C gene transcription.

The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements.

Animals↗