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Biomedical subjects

H Will

Publications and source records attributed to H Will.

At least 181 records · Page 10Linked to original sources
PubMed1988

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma.

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.

Carcinoma, Hepatocellular↗
PubMed1988

Serological evidence for expression of the polymerase gene of human hepatitis B virus in vivo.

Human sera were examined by immunoblotting for antibodies against the polymerase (reverse transcriptase) believed to be encoded in the P open reading frame (ORF) of human hepatitis B virus (HBV). Sera from patients with self-limited and chronic hepatitis reacted specifically with fusion proteins containing different domains of the P ORF. The results indicate that this ORF is expressed, and that the corresponding proteins contain at least two immunogenic domains. In contrast to human immunodeficiency virus, induction of antibodies against reverse transcriptase appears to be less common for HBV, and may depend on long persistence of infection.

Acute Disease↗
PubMed1988

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.

Amino Acid Sequence↗
PubMed1987

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum.

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.

Animals↗
PubMed1987

Attempts towards a serological diagnosis of ankylosing spondylitis.

Polytene chromosomes of salivary glands as well as nuclear proteins from Kc-cells of Drosophila melanogaster have been used as substrate to identify and evaluate the diagnostic value of crossreacting antibodies present in sera of AS patients. The diagnostic significance of the recently described anti-93D antibody (Lakomek et al., 1984) was confirmed by screening sera of patients with definite or suspected AS using cytoimmunofluorescence on the polytene chromosomes. In addition, four new antibodies could be identified in AS sera by immunoblotting. Simultaneous detection of these antibodies supports the diagnosis of AS and is most useful in diagnosis of early stages of this disease.

Adult↗
PubMed1987

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line.

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.

Amino Acid Sequence↗
PubMed1987

Expression of the hepatitis B virus core gene in vitro and in vivo.

The core gene of hepatitis B virus contains two in-phase AUG codons which may both be used in the viral life cycle. By in vitro translation of transcripts produced in vitro, we investigated the corresponding core gene products and their counterparts in vivo. Depending on the location of the 5' end of the transcripts, two major core gene-derived proteins were obtained. In transcripts with both in-phase AUGs, only the first one was efficiently used and resulted in synthesis of a 25-kilodalton protein (precore). This protein contains a leader sequence and could be cotranslationally processed to a protein of 22.3 kilodaltons. Translation of transcripts lacking the first AUG of the core gene produced a core protein of 21.5 kilodaltons which comigrated with the core antigen expressed in infected livers. These data suggest that the major nucleocapsid protein expressed in vivo is initiated at the second ATG of the C gene and that a precore protein is probably synthesized as a precursor protein which is cotranslationally processed. Proteins consistent in size with processed and unprocessed precore proteins detected in woodchuck hepatitis virus-infected livers support this conclusion.

Animals↗
PubMed1987

Replication strategy of human hepatitis B virus.

To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses.

Animals↗
PubMed1987

Characterization of calcium antagonist receptors in highly purified porcine cardiac sarcolemma.

Drug receptors for the Ca-antagonists nitrendipine and (-)desmethoxyverapamil (D-888) were studied in porcine cardiac sarcolemma. The dihydropyridine (DHP) derivative [3H]nitrendipine binds to a single population of high affinity sites (KD = 0.44 +/- 0.10 nM, Bmax = 2.43 +/- 0.19 pmol/mg; (n = 8), whereas the phenylalkylamine derivative [3H](-)D-888 interacts with high and low affinity sites of KD = 5.1 +/- 1.4 nM and KD = 438 +/- 86 nM (n = 6) in the same membrane preparations. The antagonists tested differ also with respect to their pharmacological efficacy on cultured intact heart myocytes. The EC50 value of the negative chronotropic effect exerted by D-888 corresponds to its high affinity binding component. Nitrendipine exhibit a 200-fold lower pharmacological efficacy, as would be expected from binding data.

Animals↗
PubMed1986

A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein.

A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.

Animals↗
PubMed1986

Putative reverse transcriptase intermediates of human hepatitis B virus in primary liver carcinomas.

Nucleocapsid-pol fusion proteins have been detected by serological screening hepatocellular carcinoma tissues that contain hepatitis B virus (HBV) DNA. The existence of these fusion proteins suggests that HBV may synthesize its reverse transcriptase in a fashion analogous to the way that retroviruses synthesize and process a precursor. The accumulation of HBV reverse transcriptase intermediates in tumorous tissues and not in other tissues may be related to the absence of viral core particles and possibly contributes to tumor development.

Animals↗
PubMed1986

Sarcolemmal Na-Ca exchange and sarcoplasmic reticulum calcium uptake in developing chick heart.

Ontogenetic changes in calcium transport mediated by the sarcolemmal Na-Ca exchanger and by the sarcoplasmic reticulum calcium pump were studied in crude membranes from chick heart. Transport activities were evaluated per mass of membrane protein and heart tissue. Relative to unit heart mass Na-Ca exchange activity increases linearly from embryonic day 4 to day 10 of newborn stage. The overall increase is about 20-fold. An excellent correlation exists between activity of sodium gradient-induced calcium uptake and ouabain-sensitive (Na,K)-ATPase in crude membranes of embryonic, newborn and adult hearts. In the same membrane preparations active calcium uptake into vesicles of sarcoplasmic reticulum increases about 3-fold from embryonic day 4 to embryonic day 7, and then increases continuously until day 20. This is followed by a 3-fold elevation in reticular calcium accumulation at hatching on day 21. Maximal sarcoplasmic reticulum calcium transport activity reached at day 10 after hatching is 40- to 50-fold greater than activity values at embryonic day 4. In adult hearts the activities of both Na-Ca exchange and reticular calcium uptake drop to levels characteristic for the late embryonic period. This comparative study of sarcolemmal sodium gradient-dependent calcium flux and reticular calcium sequestration demonstrates that during chick heart differentiation the two calcium transport systems do not develop in parallel. Na-Ca exchange appears to play a greater role in calcium control of embryonic as compared to newborn and adult hearts. By contrast, the contribution of sarcoplasmic reticulum calcium transport to cardiac calcium movements becomes more predominant during and after hatching.

Animals↗
PubMed1986

Putative Ca2+ channels in cardiac membranes. Subcellular distribution of [3H]nitrendipine receptors.

The binding of Ca2+ channel blocking drug nitrendipine was studied in purified sarcolemma (SL), fragmented sarcoplasmic reticulum (SR), and crude membranes isolated from porcine left ventricle. The density of specific [3H]nitrendipine binding sites was compared to Na+/Ca2+ exchange and (Na+, K+)ATPase activities of each membrane preparation. Enrichment of [3H]nitrendipine binding sites in purified SL correlates excellently with the purification of the two studied sarcolemmal marker activities. The results suggest that high affinity nitrendipine receptors are solely localized in cardiac SL.

Animals↗