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Biomedical subjects

H Hilz

Publications and source records attributed to H Hilz.

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

ADP-ribosyl-protein conjugate subclasses in various tissues. Specific influence of thyroid hormone on liver conjugates.

The amounts of endogenous mono (ADP ribose)-protein conjugates and their hydroxyl-amine-sensitive and hydroxylamine-resistant subfractions in various tissues of the mouse were determined and compared with poly (ADP-ribose) conjugates. Total mono-(ADP-ribose) conjugates did not correlate with poly (ADP-ribose) residues or the cellularity of a tissue, but were related to the protein content and the amounts of NAD+ +NADH. The hydroxylamine-resistant subfraction, which in liver is mainly associated with the mitochondria [Adamietz, Wielckens, Bredehorst, Lengyel & Hilz (1981) Biochem. Biophys. Res, Commun. 101, 96-103], did not correlate with the tissue content of cytochrome c oxidase. In hypothyroid mice hydroxylamine-resistant mono (ADP-ribose) conjugates of the liver were increased by a factor of two while the hydroxylamine-sensitive conjugates did not change significantly under these conditions. Upon administration of thyroxine the hydroxylamine-resistant subfraction returned to normal.

Adenosine Diphosphate Ribose↗
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↗
PubMed1982

Steroidogenic capacity of isolated adult mouse Leydig cells does not decrease with age.

Leydig cells were isolated from the testes of adult young (5--7 months), old (21 months), and senile (27 months) mice using a highly preservative Percoll procedure. The yield in Leydig cells per testis from young animals was slightly lower than from the old age groups. hCG-induced testosterone synthesis proceeded in a linear fashion for at least 5 h in all three groups. The maximal steroidogenic capacity of cells from old animals was identical to that from young adults. There was no significant difference between Leydig cells from young and old mice with respect to hCG-induced cAMP accumulation and protein kinase activation. Determination of hCG concentrations required for half-maximal stimulation of testosterone synthesis and cAMP accumulation showed identical, or even lower, values in the old age groups. The phenomenon may be connected with the significant augmentation with age of the DNA content per cell (polyploidization), possibly acting as a compensatory mechanism of age-induced deficiencies. Detailed kinetic studies of cAMP accumulation, protein kinase activation, protein kinase activation, and steroidogenesis as well as ultrastructural analyses support the findings of unimpaired or increased capacities of the testosterone-forming cells in old animals. Thus, the aging of Leydig cells appears to differ from that of other tissues of the mouse (e.g. skeletal muscle), which exhibit decreasing abilities to respond to stimuli.

Aging↗
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

Positive and negative adaptation of muscle enzymes in aging mice subjected to physical exercise.

CWI mice at the age of 6, 22 and 27 months were subjected to a controlled physical training program for 5 weeks. Changes in specific activity of the enzymes aldolase, superoxide dismutase and catalase of the total hind-leg muscle tissues were followed. During the training period, specific activities of these muscle enzymes exhibited an adaptive increase in the 6 and 22 months groups. In senile (27 months) mice, however, enzyme activities decreased as a consequence of the physical challenge. Total body weight was not altered under these conditions. Immunotitration of extracts with anti-aldolase and anti-creatine kinase antibodies showed no significant differences, indicating that there was no accumulation of disappearance of faulty proteins during aging and physical exercise.

Aging↗
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↗