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Biomedical subjects

E Pfaff

Publications and source records attributed to E Pfaff.

At least 73 records · Page 4Linked to original sources

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↗

Characterization of large surface proteins of hepatitis B virus by antibodies to preS-S encoded amino acids.

The major surface protein of HBV, the 226-amino-acid HBsAg, is encoded in the 3' proximal segment of the preS-S gene of 389 codons. To identify gene products from the 5' proximal preS sequence, DNA fragments from the preS region were expressed in Escherichia coli as fusion proteins. Antisera prepared against these fusions were used to screen serum proteins of HBV-infected individuals, and found to react specifically with the two large HBV surface proteins of 39 and 42 kDa. The presence of these proteins could be correlated with acute HBV infection. Analysis by Western blotting using the preS sequence-specific antisera and HBV particles separated into spheres, filaments, and Dane particles confirmed that these proteins were associated with the native virus. Dane particles containing active DNA polymerase could be immune precipitated by the preS-specific antibodies, showing that the preS-coded part of these surface proteins is located on the surface of the virion.

DNA, Viral↗

Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection.

The presence of the two "large" surface proteins of hepatitis B virus (HBV), P39 and GP42 of pre-S1-hepatitis B surface antigen, was assayed in the serum of an experimentally infected chimpanzee by using antibodies to a pre-S1-specific fusion protein synthesized in Escherichia coli. The immune response to pre-S1-hepatitis B surface antigen was monitored by using the pre-S1 fusion protein as an antigen. pre-S1 proteins were detected in the serum early in the course of infection and prevailed as long as hepatitis B surface antigen did, together with hepatitis B e antigen and viral DNA. Thus, the pre-S1 antigen can be considered a novel diagnostic marker for acute HBV infection. Antibodies to pre-S1, both immunoglobulin M and G classes, were also detected early in infection, shortly after the appearance of the pre-S1 antigen, suggesting its strong immunogenicity in vivo. The anti-pre-S1 antibodies therefore also represent an early serological marker for acute HBV infection and, owing to their early appearance and persistence, may play a role in the neutralization of the virus.

Acute Disease↗

On the state of calcium ions in isolated rat liver mitochondria. V. Development of a rapidly dischargeable pool of mitochondrial calcium during calcium-induced transition.

Ruthenium red-induced calcium efflux from rat liver mitochondria is accelerated in the course of calcium-mediated mitochondrial transition. Analysis of the efflux patterns reveals biexponential kinetics consisting of the native slow phase preceded by a faster phase developing in the presence of calcium. The data are consistent with a progressive transformation of mitochondrial calcium into a rapidly dischargeable pool prior to spontaneous calcium release. Analysis of the efflux pattern is proposed as a method to discriminate between different mechanisms of modulation of ruthenium red-induced calcium efflux. Thus, it is shown that acetate, in contrast to phosphate, stimulates ruthenium red-induced calcium efflux due to the development of the rapidly dischargeable pool of calcium.

Animals↗

Regulation of canalicular bile formation by alpha-adrenergic action and by external ATP in the isolated perfused rat liver.

In isolated perfused rat liver, addition of adrenaline induced a complex response of bile flow including rapid, reversible stimulation (1/2-2 min), reversible inhibition (2-10 min), and prolonged stimulation. Both the reversible stimulation and the inhibition were mimicked by the alpha-sympathomimetic agonist phenylephrine but not by the beta-agonist isoproterenol. The reversible stimulation was a very early effect being terminated prior to all other alpha-adrenergic responses of liver. External ATP considerably lowered bile flow while inducing release of glucose and lactate, inhibition of respiration, and a reversible efflux of Ca2+. Variations of mannitol clearance parallel to those of bile flow indicate a canalicular origin of all changes.

Adenosine Triphosphate↗

Effect of depletion of cellular glutathione on methotrexate influx, efflux and retention in hepatocytes.

In isolated hepatocytes the influence of cellular glutathione (GSH) on initial influx, net uptake and efflux of methotrexate (MTX) was determined. Endogenous glutathione in rat liver cells was depleted by either fasting of rats or by in vivo administration of phorone prior to cell preparation. The initial rate of influx of MTX was found to be higher in hepatocytes of fasted and phorone-treated rats than in those of untreated, fed control rats. The Km values for the methotrexate influx in GSH-deficient hepatocytes were up to 3 times lower than in normal cells, whereas Vmax remained unchanged. These results disclose an increased efficiency of the MTX transport system in cells with diminished cellular GSH levels. On the other hand, titration of external membrane SH groups by 203Hg p-CMBS revealed up to three times higher amounts of free SH groups on cells from starved and phorone-treated rats than on hepatocytes of fed rats. Increased efficiency of the MTX transport system in GSH-deficient cells may, therefore, be interpreted as increased capacity of the MTX transport carrier for which free membrane SH groups are known to be essential. Despite activation of initial transport of MTX here, later net accumulation of MTX became smaller than in cells with normal GSH levels. Efflux of MTX from liver cells was not influenced by fasting or phorone treatment of rats, however, the "nonexchangeable" pool of MTX was found to be decreased, which indicates inhibition of formation of MTX polyglutamates here. This inhibition was most likely responsible for the decreased amounts of MTX finally accumulated in GSH-deficient hepatocytes.

4-Chloromercuribenzenesulfonate↗

Diurnal variation of methotrexate transport and accumulation in hepatocytes--a consequence of variations in cellular glutathione.

Diurnal variation of the methotrexate (MTX) initial influx and net uptake in isolated rat liver cells was studied in dependence on diurnal variation of cellular glutathione. It was found that the most significant differences concerning the MTX initial transport and accumulation were observed between hepatocytes prepared at 1200 hr when cellular glutathione reached its maximum, and those isolated at 0000 hr when liver glutathione had its minimal concentration. The initial influx of MTX was the biggest in cells isolated at 0000 hr and the smallest in cells prepared at 1200 hr. The Km values in cells with low cellular glutathione (at 1800 and 0000 hr) were about three times smaller than in cells with high glutathione (at 0700 and at 1200 hr), whereas the Vmax value remained unchanged. Titration of external membrane SH groups by [203Hg]p-CMBS revealed a much larger amount of free SH groups on cells having low glutathione level than on those with high glutathione. Despite big initial influx of MTX found in cells with low cellular glutathione, net accumulation of MTX was significantly smaller in these cells as compared with hepatocytes having high glutathione level. In conclusion, the present studies confirmed a statement of the preceding paper on the important role of cellular glutathione played in methotrexate transport and accumulation in rat liver cells.

Animals↗

Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product.

The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.

Alpharetrovirus↗

DNA-binding activity is associated with purified myb proteins from AMV and E26 viruses and is temperature-sensitive for E26 ts mutants.

Oncogene protein products from avian myeloblastosis virus, p48v-myb, and from avian leukemia virus E26, p135gag-myb-ets, are located predominantly in the nucleus of nonproducer bone marrow cell clones, as revealed by indirect immunofluorescence. Both oncogene proteins were purified by immunoaffinity chromatography using monoclonal antibodies against p19 and immunoglobulins specific for myb, which was expressed in bacteria for antibody production. The purified proteins bind to DNA in vitro. In contrast, purified p135gag-myb-ets proteins from several mutants of E26 virus, temperature-sensitive for myeloblast transformation, either lost their abilities to bind to DNA or exhibited highly thermolabile DNA-protein interactions in vitro. DNA binding of AMV and E26 oncogene proteins is inhibited by myb-specific immunoglobulins. Our results suggest that lesions in the myb oncogene affect transformation as well as DNA binding of myb proteins in vitro.

Animals↗

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗

On the state of calcium ions in isolated rat liver mitochondria IV. Prevention of phosphate-induced mitochondrial destruction by ruthenium red-insensitive calcium release.

Ruthenium red prevented the spontaneous calcium release and the accompanying mitochondrial destruction occurring in calcium-loaded mitochondria in the presence of phosphate. Under these conditions delta pH and membrane potential delta psi were preserved and the ruthenium red-induced calcium efflux was low and at a constant rate. On prolonged incubation with calcium prior to addition of ruthenium red increasingly more mitochondrial calcium developed into a pool rapidly dischargeable by ruthenium red. This development was accompanied by stimulation of respiration which was, however, not abolished by ruthenium red as could have been expected if it had been caused by calcium cycling. Calcium therefore altered mitochondria by a different mechanism than by cycling across the inner membrane.

Animals↗

Inhibition of DNA binding of purified p55v-myc in vitro by antibodies against bacterially expressed myc protein and a synthetic peptide.

To identify viral myc proteins, we have prepared myc-specific antibodies: (i) against a synthetic peptide corresponding to the nine carboxy-terminal amino acids of the viral myc (C9); (ii) against a bacterially expressed viral myc protein obtained by inserting the SalI-BamHI fragment of the viral MC29 DNA clone in the expression vector pPLc24. Both antisera recognize a protein of 55 000 mol. wt., p55v-myc, in MH2- and OK10-transformed fibroblasts. The protein is located in the nucleus, as shown by indirect immunofluorescence and cell fractionation. Antibodies against the C9 peptide were used to purify the p55v-myc by immunoaffinity column purification (3000-fold) from OK10- and MH2-transformed fibroblasts. p55v-myc binds to double-stranded DNA in vitro as does p110gag-myc. DNA binding in vitro is inhibited by the immunoglobulin fraction of antibodies against the bacterially expressed myc protein. Furthermore, a synthetic peptide consisting of 16 amino acids (C16) was used to isolate specific immunoglobulins which also inhibit DNA binding in vitro. OK10 codes, in addition to p55v-myc, for a p200gag-pol-myc polyprotein. The majority of this protein is located in the cytoplasm (79%). The purified protein binds to single-stranded RNA in vitro, unlike other gag-myc or myc proteins.

Antibodies, Viral↗

Expression of hepatitis B antigens with a simian virus 40 vector.

Recombinant DNA molecules consisting of the simian virus 40 (SV40) early region and different subgenomic hepatitis B virus DNA fragments were constructed in vitro and packaged in vivo into SV40 capsids by using a complementing SV40 helper virus. Upon infection with these virus stocks the three known hepatitis B-specific antigens were expressed under SV40 control. The surface antigen was released into the medium, and the core antigen and its derivative hepatitis B e antigen were only detected intracellularly. Size analysis of the core gene product(s) by immunoblotting revealed the presence of a single protein species identical with the 21-kilodalton core antigen isolated from human liver. The hepatitis B core antigen expressing construct did not contain a putative precore sequence, indicating that such a sequence is not needed for hepatitis B core antigen synthesis in animal cells. S1 analysis demonstrated the use of SV40 signals for initiation and polyadenylation of the core gene transcripts. In addition, a processing-polyadenylation signal was identified within the core gene.

Cell Line↗

On the state of calcium ions in isolated rat liver mitochondria. I. Ion fluxes and volume changes upon Ca2+ uptake under various ionic conditions.

As to functional consequences of Ca2+ uptake in isolated rat liver mitochondria, we simultaneously measured 3H2O and [14C]sucrose spaces, monovalent cation distribution, membrane potential and delta pH across the inner membrane, and [32P]phosphate and 45Ca2+ content in parallel incubations of different ionic composition. Without added Ca2+ and phosphate, mitochondrial matrix volume, membrane potential, and delta pH depended on the concentration and permeability of monovalent cations. Despite large differences in membrane potential, maximal Ca2+ uptake was identical under all conditions. Ca2+ uptake never provoked a volume change from which an osmotic active state of mitochondrial Ca2+ could be concluded. If matrix volume shrunk this could be totally accounted for by the loss of alkali ions exchanging for calcium ions. Even phosphate taken up in conjunction with Ca2+ was osmotically silent. Volume increases here occurring if K+ was permeabilized, solely resulted from K+ uptake, though this condition may give rise to irreversible mitochondrial damage with Ca2+ and phosphate release. As mitochondrial Ca2+ is bound, an electro-chemical equilibrium across the membrane is impossible for this ion. This has to be considered in any model describing equilibria of Ca2+ with mitochondria, though present models neglect this state of mitochondrial Ca2+.

Animals↗

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release.

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.

Acetoacetates↗

Bile secretion in hemoglobin-free perfused rat liver.

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.

Animals↗