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H Klocker

Publications and source records attributed to H Klocker.

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

Growth-stimulating effect of adrenal androgens on the R3327 Dunning prostatic carcinoma.

Adrenal androgens are discussed as a reason for tumor progression after androgen ablation therapy. Because of the difference in the secretion of androgens by the adrenals of humans and rats, there is no reliable tumor model to study the role of adrenal androgens in tumor progression. Therefore, the main adrenal androgens were administered to rats in order to mimic human endocrine conditions. Application of dehydroepiandrosteron-sulfate (DHEA-S) alone or a mixture of androstendione (A), 11 beta-hydroxyandrostendione (OHA), dehydroepiandrosterone (DHEA), and its sulfate (DHEA-S) to castrated rats caused only a slight increase of prostate and seminal vesicle weight. Contrary to these findings, growth of the R3327 prostatic carcinoma in castrated rats was greatly stimulated by these adrenal androgens up to the level of the intact control. Thus, in spite of androgen ablation, tumor progression could be induced by exogenous adrenal androgens.

Adenocarcinoma↗
PubMed1990

Analysis of the immune-encodrine feedback loop in the avian system and its alteration in chickens with spontaneous autoimmune thyroiditis.

In both mammals and chickens, immunization with exogenous antigens results in a surge of serum glucocorticoid hormone levels concomitant with the antibody response. This effect is mediated by glucocorticoid-increasing factors (GIF) produced by cells of the immune system. In the avian system, GIF appear to act via the hypothalamo-pituitary axis and not directly on the adrenal gland. Interleukin 1 is the main active substance responsible for GIF activity, as shown by molecular sieve and immunoaffinity chromatography studies. In contrast to data from mammals, we found no evidence that interleukin 2 elevates chicken corticosterone. Obese strain chickens with spontaneous Hashimoto-like autoimmune thyroiditis are deficient in their in vivo GIF response. Because no differences were found between autoimmune and healthy chickens in the corticosterone response of the adrenal gland after ACTH administration, and since autoimmune animals are able to react normally to immobilization stress, it is assumed that this deficiency is due to a specific defect rather than a general disturbance in the endocrine system.

Adrenal Glands↗
PubMed1989

Clinical and biochemical studies in three patients with severe early infantile Cockayne syndrome.

We present clinical and biochemical data from three patients with severe Cockayne syndrome (CS) of very early onset. Unlike in classic CS, signs became evident in the first weeks of life and led to unusually early death. Fibroblasts from two of the patients showed a complete defect of the repair of UV-induced thymine dimer lesions. They were unable to remove thymine dimer lesions from their DNA, had a severe reduction of the RNA synthesis rates after UV irradiation, and showed no reactivation of an UV-inactivated indicator gene and no DNA recondensation after UV irradiation. DNA repair investigated in these two fibroblast cell strains resembled that of xeroderma pigmentosum cells of complementation group A. In contrast, fibroblasts from the third patient showed the same in vitro repair characteristics as classic CS cells.

Cell Survival↗
PubMed1988

Decreased level of thymidine in the serum of obese strain (OS) chickens with spontaneous autoimmune thyroiditis.

We have previously shown that normal chicken serum (NCS) is able to interfere with the IL 2 promoted incorporation of DNA precursors into T lymphoblasts and that serum derived from autoimmunity prone Obese strain (OS) chickens is deficient in this respect. This "defect in non-specific suppression" has been speculated to be one of the causes for T cell hyperreactivity in the OS. In this study we present several lines of evidence that the suppressive effect of normal chicken serum (NCS) on 5-(125Iodo)-2-deoxyuridine (125IUdR) uptake into chicken T blasts is a competition artefact due to cold thymidine (TdR) present in NCS. Inhibition of 125IUdR required the continuous presence of NCS and suppression of 3H-TdR incorporation could be competed for by increasing the dose of the radiolabel. Molecular sieve chromatography followed by reversed phase high performance liquid chromotagraphy revealed the "inhibitory" activity to co-elute with TdR. Moreover, NCS did not suppress protein synthesis by chicken T cells growing with IL 2 and did not affect oxidative metabolism, cell viability, expression of IL 2 receptors, or percentages of cells in the S phase of the cell cycle. In accordance with these data, OS-sera suprisingly contain less TdR than those from normal controls. Experiments involving crosses of the OS with the normal inbred CB strain, revealed that the subnormal serum TdR level of the OS is an autosomally dominant trait which, however, segregates from T cell hyperreactivity. These findings falsify our previous hypothesis that a defect in specific IL 2 antagonists might be involved in T cell hyperfunction of the OS and indicate that NCS is devoid of factors which neutralize IL 2 function.

Animals↗
PubMed1987

ADP-ribosyltransferase from Helix pomatia. Purification and characterization.

ADP-ribosyltransferases from several higher eukaryotes have been purified and characterized, but little is known about ADP-ribosyltransferases in lower eukaryotes. We have purified an ADP-ribosyltransferase (EC 2.4.2.30) from Helix pomatia. The enzyme has an apparent Km of 26.7 microM. Optimal conditions for the enzyme reaction are 17.5 degrees C and pH 8. The time course is linear during the first 10 min of the reaction. The enzyme is capable of poly-ADP-ribosylation. The most highly purified preparation shows one major band at an Mr of 75,000 on electrophoresis in an SDS/polyacrylamide gel, with minor bands at Mr 115,000 and 155,000. Re-activation of SDS/polyacrylamide gels in situ shows the 75,000-Mr band to be enzymically active and additional active bands with Mr values of 115,000, 90,000 and 87,000 respectively. The 115,000-Mr and 75,000-Mr bands cross-react with a polyclonal affinity-purified antiserum against human ADP-ribosyltransferase. Like enzymes from higher eukaryotes, the activity from Helix pomatia is inhibited by thymidine, theophylline, theobromine nicotinamide, 3-methoxybenzamide and 3-aminobenzamide, and is dependent on histone and DNA.

Animals↗
PubMed1987

ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme.

Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11,700-fold purified activity shows a major protein band at a molecular mass of 75,000 kDa in a SDS-polyacrylamide gel. In situ reactivation of SDS gels showed the 75,000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115,000, 90,000 and 87,000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparent Km for NAD+ of 24 microM. The temperature optimum is 10 degrees C compared with 25 degrees C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout.

Animals↗
PubMed1987

Measurement of glycated protein by a rapid and specific method for absolute quantification of lysine-bound glucose.

We modified the liquid-chromatographic assay of Schleicher and Wieland (J Clin Chem Clin Biochem 1981; 19:81-7) for measuring lysine-bound glucose in serum proteins, increasing its performance and practicality. After precipitating serum proteins from 10- to 50-microL samples with ethanol (700 mL/L) and hydrolyzing these in 6 mol/L HCl, we inject 20 microL of the diluted hydrolysate directly into the chromatograph, which consists of an acid-resistant C18 precolumn combined with a high-resolution C18 main column. The eluent is 3.5 mmol/L H3PO4 solution containing 30 mL of acetonitrile per liter. These modifications increase sensitivity, provide excellent resolution and longevity of stationary phases, shorten assay times to 15 to 20 min, and are suited for automation. The assay is highly sensitive and highly specific, quantifying nanomoles of lysine-bound glucose per milligram of protein. A precision (CV) of 5.1% is achievable at physiological and supra-physiological glucose concentrations, and analytical recovery is 99%. This inexpensive method has been applied to serum albumin, bulk serum proteins, and preparations of low-density lipoproteins and immunoglobulins.

Blood Proteins↗
PubMed1987

Isolation of a cDNA clone for human NAD+: protein ADP-ribosyltransferase.

NAD+:Protein ADP-ribosyltransferase (EC 2.4.2.30) (ADPRT) was purified from human placenta by affinity chromatography. With the purified enzyme specific antibodies were raised and partial amino acid sequences were determined. To one of the amino acid sequences corresponding oligonucleotides were synthesized. A sized HeLa lambda gt11 cDNA library was constructed and screened. Positive clones were characterized to be ADPRT specific by immuno- and hybridization techniques. Clone ADPRT-G8 reacted with affinity chromatographically purified specific antibodies and with two specific oligonucleotides. The DNA of this clone detected an mRNA of about 4 kb, sufficient in size to code for the ADPRT with an Mr of 116,000. Partial sequence analysis of this clone confirmed its identity by revealing sequences which code for peptides which were found in cyanogen bromide (CNBr) fragments of the purified enzyme. The ADPRT-G8 clone was characterized with respect to its restriction pattern. The cloned ADPRT cDNA now opens the possibility to investigate the role of this enzyme in control of cellular functions.

ADP Ribose Transferases↗
PubMed1986

Isolation of ADP-ribosyltransferase by affinity chromatography.

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.

Animals↗
PubMed1986

Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription.

Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.

Alkylating Agents↗
PubMed1985

Fibroblasts from patients with Fanconi's anemia are not deficient in excision of thymine dimer.

Fibroblasts from a patient with Fanconi's anemia were reported to show a defective excision of pyrimidine dimer [15]. We developed a sensitive radioimmuno assay which is specific for thymine dimer, the main ultraviolet photoproduct, and reinvestigated the thymine dimer excision in fibroblasts from patients with Fanconi's anemia. The analysis of 7 Fanconi's anemia cell lines did not agree with the claim mentioned above that was derived from only one Fanconi's anemia cell line. All cell lines we studied, including the cell line used previously [15], excised thymine dimer from their DNA with excision rates similar to those of normal fibroblasts. Additionally, in two Fanconi's anemia and in two normal fibroblast cell lines the repair capacity was examined.

Adult↗
PubMed1984

A synthetic hapten for induction of thymine-dimer-specific antibodies.

High specificity and sensitivity of thymine cyclobutane dimer (thy[]thy) detection were obtained by a radioimmunoassay. Attempts to raise thy[]thy-monospecific antibodies with antigens produced according to conventional methods were unsuccessful. Thy[]thy-specific antibodies could only be raised by using a new strategy to bind thy[]thy to protein: thymine was activated by trimethylsilylation and alkylated at N1 yielding N1-thyminebutanoic acid which was dimerised by ultraviolet treatment. The resulting derivative of thymine cyclobutane dimer was coupled to bovine serum albumin by the active-ester method. The new strategy appears to be generally applicable for binding haptens, such as DNA bases, photoproducts etc, to proteins via a derivative containing a carboxyl group. Immunisation of rabbits with the thy[]thy-bovine-serum-albumin conjugate prepared by the new method resulted in a highly specific antiserum which allows detection of thy[]thy down to 0.06 p mol (15pg). The thy[]thy-specific radioimmunoassay was applied to measure thy[]thy formed in human fibroblasts which were exposed to sunlight at altitudes of 600 m or 2300 m. The amounts of thy[]thy formed in an hour corresponded to doses of 14 J m-2 and 24 J m-2, respectively, of an ultraviolet light lamp emitting predominantly 245-nm light.

Animals↗
PubMed1983

DNA repair dependent NAD+ metabolism is impaired in cells from patients with Fanconi's anemia.

In vitro cultivated fibroblasts derived either from patients with Fanconi's anemia (FA) or from healthy probands were analyzed for their DNA repair-dependent NAD+ metabolism. No difference in NAD+ pools was found. NAD+ consumption after cell damage by u.v. irradiation was, however, significantly reduced in FA cells. Several FA cell lines had a lowered ability to transfer ADP-ribose to acid-precipitable material. Additionally, a decreased activity of NAD: protein ADP-ribosyltransferase was found for three FA cell lines. Our data indicate, that FA is accompanied by a defective NAD+ metabolism during DNA repair.

Cell Extracts↗
PubMed1982

Intracellular distribution of DNA topoisomerase I in fibroblasts from patients with Fanconi's anaemia.

The activity of DNA topoisomerase I(DNA nicking-closing enzyme) was analysed in cytoplasmic and nuclear extracts of six independently derived Fanconi and four normal fibroblast cell lines. In all experiments the total cellular activity was predominantly found in the nuclear extracts (88-100%). In addition, a minor proportion of the enzyme (up to 12%) was randomly present in some of the cytoplasmic fractions of both Fanconi and normal fibroblasts. These results indicate that Fanconi's anaemia is probably not due to or accompanied by a maldistribution of topoisomerase I between nuclei and cytoplasm.

Anemia, Aplastic↗
PubMed1982

Repair rate in human fibroblasts measured by thymine dimer excorporation.

The UV photoproduct, thymine dimer (-TT), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m2 creates 0.045% -TT (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of -TT/T is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m2. The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.

Cells, Cultured↗