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R Bredehorst

Publications and source records attributed to R Bredehorst.

At least 55 records · Page 3Linked to original sources
PubMed1986

Immobilized doxorubicin increases the complement susceptibility of human melanoma cells by protecting complement component C3b against inactivation.

Human melanoma cells resistant to killing by monoclonal antibody R24 plus human complement became susceptible after treatment with doxorubicin (adriamycin). Treatment with doxorubicin prevented the rapid degradation of surface-bound complement component C3b that has been identified as a protective mechanism of complement-resistant melanoma cells. Doxorubicin caused the increased complement susceptibility as free drug and after immobilization onto glass beads to prevent cellular uptake. Immobilized doxorubicin was more effective than free drug, causing enhanced complement susceptibility at concentrations where the free drug was no longer active. In contrast to free doxorubicin, which exhibited a direct cytotoxic effect leading to cell death within 4 days, immobilized doxorubicin did not affect cell viability. These findings suggest that combination therapy of the complement-activating monoclonal antibody R24 with the complement-enhancing drug doxorubicin may be a promising approach for the treatment of melanoma.

Antibodies, Monoclonal↗
PubMed1985

Quantitation of hepatitis B virus (HBV) core antigen in serum in the presence of antibodies to HBV core antigen: comparison with assays of serum HBV DNA, DNA polymerase, and HBV e antigen.

A quantitation procedure for hepatitis B core antigen (HBcAg) in serum without prior removal of antibodies to HBcAg is described. The virus nucleoprotein core was released from hepatitis B virus (HBV) particles by treatment with Nonidet P-40 detergent and allowed to form immune complexes with homologous antibodies to HBcAg present in the sera of HBV-infected individuals. After precipitation with 2.0% polyethylene glycol-1.5% Tween 20, the HBcAg immune complexes were dissociated by treatment with 3 M KSCN and then adsorbed onto polystyrene beads in the presence of the SCN- ions. Thereby, HBcAg and antibodies to HBcAg were linked independently of each other to the matrix, and the core antigen could be quantitated directly by incubation of the beads with 125I-labeled anti-HBc. Even in the presence of an excess of antibodies to HBcAg in the polyethylene glycol precipitates, HBcAg could be detected without appreciably affecting the sensitivity. The assay proved to be specific for core determinants and exhibited excellent reproducibility. The application of the HBcAg assay in 185 hepatitis B e antigen-positive sera revealed HBc antigenemia in 99% of the sera containing hepatitis B e antigen at titers of greater than or equal to 1:256 and 43% of the sera with lower hepatitis B e antigen levels. However, only in 6 of the 34 HBcAg-negative sera could HBV DNA be detected by blot hybridization. When correlated with HBV-associated DNA polymerase (DNAP) activity, HBc antigenemia was found in all DNAP-positive sera (n = 95) and in 39% of the hepatitis B e antigen-positive sera without detectable DNAP activity (n = 44). Of the DNAP-negative sera with HBc antigenemia, 94% contained HBV DNA, whereas in the absence of HBcAg, HBV DNA could be detected only in 3 of 27 DNAP-negative sera. With regard to sensitivity, the HBcAg assay appeared to be less sensitive than the hybridization technique, but more sensitive than the DNAP assay.

Antigen-Antibody Complex↗
PubMed1983

Quantification without purification of blood and tissue adenosine by radioimmunoassay.

Highly specific anti-adenosine antibodies were produced in rabbits by the injection of N6-carboxymethyl adenosine-methylated serum albumin conjugates. They were used to develop a radioimmunoassay allowing the quantitation of adenosine in the range 0.1-10 pmol per sample. Inosine did not interfere except at 300 times higher concentrations, while AMP (ATP) did not displace the [3H]adenosine tracer even at 10(5) (10(6) ) times higher amounts. Due to the high specificity of the anti-adenosine antibodies, determination of blood and tissue adenosine levels could be performed directly from perchloric acid extracts. Values for human peripheral venous blood from various donors obtained with this procedure varied between 46 and 148 pmol/ml blood. The procedure was also applied to HeLa cultures with low and high intracellular adenosine. The reliability of the method was demonstrated by comparative analyses using HPLC purification of adenosine prior to the radioimmunoassay.

Adenosine↗
PubMed1983

Functional aspects of mono- and poly(ADP-ribosyl)ation: subcellular distribution and ADP-ribosyl turnover under conditions of repair and 'starvation'.

Three types of ADP-ribosyl proteins (poly(ADP-ribose) conjugates, NH2OH sensitive and NH2OH resistant mono(ADPR) conjugates) could be found in all eukaryotic cells so far studied. They changed independently under various conditions and showed an uneven subcellular distribution suggesting independent functions. Treatment of Ehrlich ascites tumor (EAT) cells with monofunctional or cross-linking alkylating agents led to rapid fragmentation of DNA and depletion of NAD while poly(ADPR) polymerase activity showed a retarded increase. Endogenous amounts of poly(ADPR) groups increased 4- to 30-fold, depending on dose, with the same initial kinetics as the loss of NAD and the appearance of DNA strand breaks. Turnover of poly(ADPR) was determined from the decay rate of the polymer after the addition of benzamide to alkylated cells. At peak elevation of poly(ADPR), an apparent half-life of about 1 min was obtained (control cells: t/2 much greater than 3 hr). There was also an accumulation of nuclear mono(ADPR) conjugates with a half-life of about 10 min. In contrast to in vitro experiments, histone H1 in vivo proved to be only a minor acceptor of ADPR groups in rat liver and in hepatoma cells. It carried less than 0.2% of total monomeric, and less than 2% of total polymeric ADPR residues. Alkylation of cells increased mono(ADP-ribosyl)ation of histone H1 to a much higher degree than poly(ADP-ribosyl)ation. Addition of benzamide to alkylated cells inhibited poly(ADPR) formation and NAD depletion, but interfered with neither DNA fragmentation nor with DNA resealing. Nevertheless, benzamide was a very effective co-cytostatic.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1983

[Results of active preventive vaccination against hepatitis B with a German vaccine].

53 persons at high risk for hepatitis B infection have been vaccinated with a hepatitis B vaccine produced in the Hygiene Institute of the University of Göttingen. The vaccine proved to be without side effects in all vaccinees. Only minor complaints at the site of inoculation were registered. Three vaccinations are necessary. Protective antibodies were present in 96% of the vaccinees after the third inoculation. The female vaccinees developed anti HBs earlier than the males, moreover they showed significantly higher antibody titers. A simultaneous active and passive immunization is possible. To minimize the risk of contracting a hepatitis B or a non A- non B hepatitis as a result of the vaccination only such vaccines should be used which have been prepared from sera containing anti-HBe and which have been inactivated with a high dose of formaldehyde.

Adult↗
PubMed1982

DNA fragmentation and NAD depletion. Their relation to the turnover of endogenous mono(ADP-ribosyl) and poly(ADP-ribosyl) proteins.

Treatment of Ehrlich ascites tumor cells with the trifunctional alkylating agent 2,3-5-tris(ethyleneimino)benzoquinone-1,4 (triaziquonum) led to rapid fragmentation of DNA and depletion of NAD while poly(ADP-ribose) synthetase activity showed a retarded increase. Poly(ADP-ribosyl) residues in treated cells increased 4- to 30-fold, but transiently, and in a dose-dependent manner, exhibiting the same initial kinetics as the loss of NAD and the appearance of DNA strand breaks when determined by the nucleoid method. Although the amounts of "activated ADP-ribosyl" groups present in the substrate NAD (80 nmol/10(8) cells) exceeded by far basal and triaziquonum-induced poly(ADP-ribosyl) groups (up to 250 pmol/10(8) cells), accelerated formation of the polymer, nevertheless, may explain at least partially the loss of NAD seen under these conditions. Addition of benzamide, a potent inhibitor of poly(ADP-ribose) synthetase, to triaziquonum-treated cells effected an immediate drop of poly(ADP-ribose) to basal values. The data indicate a biphasic decay, the half-life of greater than 85% of the polymeric ADP-ribosyl groups exhibiting a t1/2 less than 1 min under these conditions, while the residual fraction died away with t1/2 approximately 6 min. Treatment with the DNA fragmenting agent also led to a 9-fold increase of nuclear mono(ADP-ribosyl) groups, while cytoplasmic mono(ADP-ribosyl) protein conjugates were not significantly affected. The apparent half-life of nuclear mono (ADP-ribosyl) protein conjugates (8-10 min) at peak elevation was definitely longer than that of poly(ADP-ribosyl) residues. This result is consistent with the interpretation that accumulation of mono(ADP-ribosyl) groups is due to a retarded removal of the primary ADP-ribosyl group from the acceptor protein by a separate mono(ADP-ribosyl) protein glycohydrolase, being the rate-limiting step in the overall turnover of poly(ADP-ribosyl) residues.

ADP Ribose Transferases↗
PubMed1982

Mono ADP-ribosylation and poly ADP-ribosylation of proteins in normal and malignant tissues.

Three subclasses of (ADPR)n protein conjugates were quantified from intact tissue; proteins carrying poly(ADPR) and two types of mono(ADPR) protein conjugates, one susceptible, the other resistant to neutral hydroxylamine. Mono(ADPR) conjugates were found in all major compartments of the liver cell although the two subfractions were unevenly distributed. Poly(ADPR) protein conjugates appear to be restricted to the nucleus. Independent changes of the subclasses in normal and malignant tissues associated with cell growth and differentiation also point to independent functions. Hydroxylamine-resistant mono(ADPR) protein conjugates of various tissues changed with the degree of terminal differentiation. Formation of poly(ADPR) proteins, on the other hand, was stimulated by treatment of cells with alkylating agents which lead to DNA-fragmentation. This points to an involvement of polyADP-ribosylation in DNA excision repair.

Adenosine Diphosphate Ribose↗
PubMed1981

Mono- and poly-ADP-ribosylation of proteins in mouse kidney after castration and testosterone treatment.

Protein-bound mono(ADP-ribose) and poly(ADP-ribose) residues were determined in mouse kidney after castration and testosterone substitution. After these treatments, the mouse kidney undergoes significant alterations in the extent and pattern of transcription without changes in the amount of DNA and nuclear protein. The amount of mono(ADP-ribose)--protein conjugates (the hydroxylamine-sensitive and -resistant subfractions) decreased by 40% after castration, and returned to normal within 1 week after daily testosterone injections. Polymeric ADP-ribose residues, which amounted to less than 0.3% of the total protein-bound monomeric ADP-ribose, increased after castration and rapidly decreased on testosterone administration. The magnitude of these effects indicates that the decrease in mono(ADP-ribose) was not caused by a shift of monomeric residues into the polymer form. Nuclear ADP-ribosyltransferase activity showed a retarded decrease after castration, reaching 60% of the control value by day 20. After testosterone injections, enzyme activity rose to normal within 3-4 days. The amounts of the substrate NAD+ as well as of NAD+ + NADH also declined after castration, and rapidly returned to values slightly above normal when the androgen was substituted. The differential response of monomeric and polymeric ADP-ribose residues to castration and testosterone treatment suggests that the two modifications serve different functions.

ADP Ribose Transferases↗
PubMed1981

Determination of 5-AMP in the presence of excess 3'(2')-AMP with the aid of antibodies raised against n6-carboxymethyl-5'-AMP conjugates. Use for the quantitation of pyridine nucleotides and of protein-bound ADP-ribose.

5'-AMP antigens were synthesized by conjugation of N6-carboxymethyl-5'-AMP (Cm65'-AMP) to native or methylated serum albumin. Injection of the antigens resulted in antibodies with high affinity and specificity for 5'-AMP in all animals, thus allowing discrimination against 3'(2')-AMP even when present at 10(4)-10(5) times higher concentrations. This specificity was comparable to that of anti 5'-AMP antibodies raised against Cm65'-AMP serum albumin antigens formed in situ from Cm6ADP-ribose serum albumin conjugates by intracellular or pericellular phosphodiesterases. The hapten in the Cm65'-AMP-methylated serum albumin conjugate appeared to be bound almost exclusively via the N6-position. Due to the free exposure of the 5'-phosphate group in this antigen, the resulting antibodies discriminated 5'-AMP derivatives substituted at the phosphate group more efficiently than derivatives with modifications in the adenine ring. It also led to the concomitant formation of adenosine-specific antibodies due presumably to dephosphorylation of the antigen by phosphatases present in the recipient animals. The conjugates formed from Cm65'-AMP and native serum albumin, which appeared to be linked to a large extent via carboxyl groups of the protein and hydroxyl groups of the ribose, recognized modifications in the adenine ring much better than substitutions at the phosphate group. However, in spite of these relatively small differences in specificity, all three types of antibodies could be used successfully to quantitate by radioimmunoassay protein-bound ADP-ribose in adult rat liver and NAD+-NADH in Ehrlich ascites tumor cells as shown by the excellent agreement of the values obtained with the three antisera.

Adenosine Diphosphate Ribose↗
PubMed1981

Mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation of proteins in developing liver and in hepatomas: relation of conjugate subfractions to metabolic competence and proliferation rates.

Endogenous levels of mono(ADP-ribose)-protein conjugates are low in fetal liver. They increase during development reaching 30-times higher levels in the adult stage. Undifferentiated hepatomas also exhibit low degrees of mono(ADP-ribosyl)ation compared with differentiated tumors. The observed changes cannot be explained by depolymerisation of pre-existing protein-bound poly(ADP-ribose) groups or elongation of monomeric ADP-ribose residues since the monomeric and polymeric residues change independently, the absolute levels of residues present in the form of polymers being 20--350-times lower than monomeric ADP-ribose residues. Subfractionation of the mono(ADP-ribose)-protein conjugates on the basis of their NH2OH sensitivity also showed independent changes during liver development. The level of the NH2OH-sensitive conjugates exhibit an inverse relationship to cell proliferation rates in normal and malignant hepatic tissues, while the NH2OH-resistant subfraction, which was hardly detectable in fetal liver, could be related to the degree of terminal differentiation (relative to adult liver). The ratio of NH2OH-resistant to NH2OH-sensitive mono(ADP-ribose)-protein conjugates being near unity in adult liver, fell to extremely low values in fetal and neonatal liver. In undifferentiated hepatomas (proliferating or stationary), however, the ratio was higher than in the adult normal tissue. This parameter, then, allows one to discriminate between malignant and normal hepatic tissues with similar proliferative capacity and similar metabolic competence. On the basis of the findings presented it is suggested that covalent modification of proteins by mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation serve multiple and independent functions.

Adenosine Diphosphate Ribose↗
PubMed1981

Protein-bound polymeric and monomeric ADP-ribose residues in hepatic tissues. Comparative analyses using a new procedure for the quantification of poly(ADP-ribose).

Determination of poly(ADP-ribose) levels was performed by a new procedure involving covalent chromatography of released polymer, degradation to phosphoribosyl AMP and quantification of this specific derivative by a radioimmunoassay. In adult rat liver, about 85 pmol polymeric ADP-ribose residues/g tissue was found. Similar values were obtained when the determination was carried out by an independent procedure not involving boronate chromatography or sedimentation of DNA. In adult rat liver, polymeric ADP-ribose residues amounted to about 1/200 of total monomeric ADP-ribose residues. Most of the polymeric ADP-ribose residues were linked to proteins by NH2OH-sensitive bonds, while mono(ADP-ribose)-protein conjugates consisted of about equal amounts of NH2OH-sensitive and NH2OH-resistant subfractions. Poly(ADP-ribose) levels immediately after birth were similar to the adult status. They decreased, however, by a factor of three at the time of most rapid post-natal liver growth (day 17). A comparison with the protein-bound monomeric ADP-ribose residues indicated independent changes, and therefore presumably independent functions of these monomeric ADP-ribose residues.

Adenosine Diphosphate Ribose↗
PubMed1980

Protein-bound mono(ADP-ribose) residues in differentiating cells of Dictyostelium discoideum.

Changes in protein-bound mono(ADP-ribose) residues during development of Dictyostelium discoideum were determined. NAD(H) levels and the amounts of the NH2OH resistant and sensitive subfractions of mono(ADPR) were found not to be different between exponentially growing and aggregation-competent cells in which mitosis had ceased. Divergent changes occurred at the differentiation stages following aggregation as indicated by an increase in the ratio of these subfractions from approx. 1 during the growth plase and aggregation competent stage to 2 in the grex, a stage which follows cell aggregation. The fraction of NH2OH sensitive conjugates closely followed the changes in total cellular protein, while the NH2OH resistant ADPR protein conjugates, when based on protein, increased during the stages following aggregation. NAD(H) and NADP(H) levels per unit DNA decreased significantly during this period. The mono(ADPR) to DNA ratio in D. discoideum is comparable to that in proliferating Physarum polycephalum and to non-proliferating adult rat liver. The total amount of mono(ADPR) residues per nucleus is, however, approximately 70-fold higher in the liver, indicating that the quantity of mono(ADPR) residues is more closely related to the size of the eukaryotic genome than to cell proliferation.

Adenosine Diphosphate Ribose↗
PubMed1980

Increase of mono(ADP-ribose) protein conjugate levels in rat liver induced by nicotinamide administration.

Protein-bound mono(ADP-ribose) residues were quantitated in the livers of nicotinamide-treated and control rats. Nicotinamide administration led to a rise in NAD+ (2.5-fold) and in protein-bound mono (ADP-ribose) residues (1.5-fold). This increase was higher in the NH2OH-sensitive mono(ADP-ribose) protein conjugates than in the NH2OH-resistant subfraction. NADH, NADP and NADPH levels did not change significantly under these conditions. The findings show that nicotinamide induced an increased ADP ribosylation of new acceptor sites, and not merely an elongation of pre-existing (ADP-ribose)n chains. A correlation of the NAD+ concentration and the extent of post-synthetic modification of nuclear proteins by mono(ADP-ribose) residues was not restricted to nicotinamide-induced changes. It was also seen in various tissues with widely differing NAD+ levels.

Adenosine Diphosphate Ribose↗
PubMed1979

Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract.

The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 degrees C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1--10 pmol.

Adenosine Monophosphate↗
PubMed1979

Intrinsic ADP-ribose transferase activity versus levels of mono(adp-ribose)protein conjugates in proliferating Ehrlich ascites tumor cells.

Transition of proliferating Ehrlich ascites tumor cells (3 days after transplantation) to the non-proliferating status (8--14 days after transplantation) was associated with an increase in total mono (ADP-ribose) protein conjugates. This increase was largely confined to the NH2OH-resistant subfraction. When the amounts of mono-(ADP-ribose) conjugates from 20% trichloroacetic acid precipitates were compared with those from 5% perchloric acid precipitates, no significant differences were seen. This fact excludes histone H1 as a major mono (ADP-ribose) acceptor in vivo in these cells. Transition to the resting state was also associated with a small decrease in NAD levels, and with no significant changes of total ADP-ribose transferase activity. However intrinsic ADP-ribose transferase activity as expressed in permeabilized cells was increased, being correlated with the changes in the level of the NH2OH-resistant mono (ADP-ribose) protein conjugates. This shows that alterations in intrinsic transferase activity may, in general, indicate similar alterations in major subfractions of ADP-ribose conjugates. Intrinsic ADP-ribose transferase activity exhibited an inverse relationship to ornithine decarboxylase activity.

Adenosine Diphosphate Ribose↗