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W Kunz

Publications and source records attributed to W Kunz.

At least 55 records · Page 3Linked to original sources
PubMed1992

Multiple nucleic acid labeling and rainbow detection.

A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described. Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids. Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate. Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane. We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening. Advantages and limitations of the method, as well as further applications, are discussed.

Alkaline Phosphatase↗
PubMed1992

Ferritins of Schistosoma mansoni: sequence comparison and expression in female and male worms.

Recombinant clones of Schistosoma mansoni cDNA libraries containing the complete coding regions of 2 different ferritin subunits have been isolated and sequenced. This allows for the first time a comparison of ferritin sequences from an invertebrate with those of vertebrates. The deduced amino acid sequences of both Schistosoma ferritin subunit clones show significant homology to vertebrate ferritin H chains. Similarity exceeds 50% identity and includes the recently identified ferroxidase center which is present only in H chains. However, non-conservative substitutions of amino acid residues lining the 3-fold symmetry channel were found, and a gap of 3 successive amino acids unique to the 2 Schistosoma ferritin sequences was identified. Remarkably, for each of the 2 genes, we found a conspicuous difference in the amount of ferritin transcripts between females and males: one of the genes is preferentially expressed in females, the other in males.

Amino Acid Sequence↗
PubMed1991

Effect of orotic acid on the generation of reactive oxygen and on lipid peroxidation in rat liver.

The pyrimidine precursor orotic acid (OA) is a constituent of dairy products and therapeutic drugs. Several recent publications point towards a tumor promoting activity of OA in rat liver. An increased production of reactive oxygen has been discussed as a possible mechanism, leading to lipid peroxidation and DNA single strand breaks. In view of contradictory results, this postulated prooxidative action of OA was reexamined with new experimental techniques. Weanling Sprague-Dawley rats were fed 1% OA in different diets for 4-35 days. The NADPH-mediated lipid peroxidation in liver homogenate and microsomes was determined in vitro by analysis of low-level chemiluminescence (CL) and the strongly correlated formation of malondialdehyde (MDA). In no case did treatment with OA result in an increase of lipid peroxidation in vitro nor did such treatment enhance the generation of reactive oxygen as measured by lucigenin CL. In accordance, the total cytochrome P-450 content as well as the activity of individual P-450 isoenzymes were unchanged. Treatment with OA did not elevate the MDA content of fresh liver homogenate when butylated hydroxytoluene (BHT) was present in the test system. However, when the antioxidant was omitted, increased levels of thiobarbituric acid reactive material were found which correlated with the triglyceride content. This could explain some published data that have been taken as indication for a prooxidative action of OA. Evidence against an increased lipid peroxidation in vivo is given by the analysis of ethane exhalation. Furthermore, no increase in DNA single strand breaks by OA treatment could be observed by the alkaline elution technique. These results do not support the hypothesis of a prooxidative activity of OA. The observed reversible decrease of the GSH/GSSG ratio is assumed to result from the reduced size of the phosphopyridine nucleotide pool due to purine deficiency and an increased consumption of NADPH by the enhanced reductive degradation of pyrimidines.

Administration, Oral↗
PubMed1990

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1990

Phenobarbital enhances the formation of reactive oxygen in neoplastic rat liver nodules.

The effect of treatment of rats with the liver monooxygenase inducer phenobarbital on the formation of reactive oxygen in neoplastic liver nodules and the surrounding normal tissue was investigated. Liver nodules were induced by treatment of rats with diethylnitrosamine (single i.p. injection of 0.15 mumol/kg body weight on day 1 after birth) followed by chronic administration of phenobarbital-sodium (PB; 0.05% in diet) after weaning. Groups of rats were kept on PB until sacrifice or were withdrawn from the promoter 3-6 weeks prior to killing. Emission of chemiluminescence was used as a sensitive means to detect the formation of reactive oxygen in microsomal preparations from the various tissues incubated with NADPH and the chemiluminigenic detector lucigenin. In addition, a 2-dimensional photon counting system has been developed that permits the analysis of the spatial distribution of lucigenin-chemiluminigenic signals over liver tissue sections incubated with reduced phosphopyridine dinucleotides. In general, we observed increased levels of reactive oxygen formation in liver nodules when compared with the normal liver tissue. Highest levels were seen in nodules that stemmed from PB-induced rats. Studies on the expression and activity of cytochrome P-450 in liver nodules as well as experiments with specific inhibitors point towards a participation of the liver monooxygenase system in reactive oxygen formation, although additional metabolic pathways seem to be involved as well. The observed increases in reactive oxygen in liver nodules of PB-treated rats might be related to the promoting activity of this drug.

Acridines↗
PubMed1989

Long-chain fatty acids act as protonophoric uncouplers of oxidative phosphorylation in rat liver mitochondria.

The effect of long-chain fatty acids (LCFA) on respiration and transmembrane potential (delta psi) in the resting state, and the rate of delta psi dissipation [d delta psi/dt)i) was investigated with oligomycin-inhibited rat liver mitochondria using succinate (plus rotenone) as substrate. The results obtained were compared with those of classical protonophores such as 2,4-dinitrophenol (DNP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB). The effects of oleate or palmitate and that of DNP or TTFB on respiration and delta psi can be described by a common force-flow relationship. These facts all in all are not compatible with a decoupler-type uncoupling mechanism of LCFA; still, they indicate that the latter are protonophores. Moreover, the oleate-induced increase in the rate of delta psi dissipation closely correlates with that in respiration, suggesting that the uncoupling activity and the protonophoric activity of LCFA are interrelated. Carboxyatractyloside (CAT) exerted only a small inhibitory effect on oleate-induced respiration and delta psi dissipation, indicating that the adenine nucleotide translocase contributes to the uncoupling effect of LCFA to a minor extent only. Proton transport through the lipid region of the membrane as mediated by permeation of the protonated and deprotonated forms of LCFA is interpreted as the main process of the uncoupling of LCFA.

2,4-Dinitrophenol↗
PubMed1989

Heterogeneity of enzyme-altered foci in rat liver.

Enzyme-altered foci (EAF) in liver are assumed to be precursor lesions for tumors in this organ. Results obtained with selected hepatocarcinogens which produce lesions of differing phenotype and growth behavior indicate that not the total number of enzyme-altered cells but rather the proliferation of individual cell clones is of major importance for additional changes leading to malignancy. Analyses including multiple marker enzymes demonstrate a relationship between foci phenotype and proliferation. The inducibility of certain downregulated enzymes in EAF indirectly suggests disturbances in the expression of regulatory genes such as proto-oncogenes. Data on Ha-ras and c-myc proto-oncogene expression in EAF are presented.

Adenosine Triphosphatases↗
PubMed1988

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids.

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the 'efficiency' of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a 'safety valve' preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.

Acetates↗
PubMed1988

Kinetic limitations in the overall reaction of mitochondrial oxidative phosphorylation accounting for flux-dependent changes in the apparent delta GexP/delta mu H+ ratio.

Changes in J0, delta muH+ and delta GexP were investigated as a function of load. The flux control coefficients, particularly those of the adenine nucleotide translocator and H+-ATPase at the maximum rate of oxidative phosphorylation were seen to strongly depend on the phosphate concentration accounting in common for the highest share in flux control. There was no unique relationship observed between JP and delta muH+ in load-controlled, well coupled systems, but JP was found to depend on delta muH+ at excessive load and increasing proton leakage. All the results presented can be elucidated on the grounds of delocalized chemiosmotic coupling.

Animals↗
PubMed1988

Metabolites of 5-fluorouracil in plasma and urine, as monitored by 19F nuclear magnetic resonance spectroscopy, for patients receiving chemotherapy with or without methotrexate pretreatment.

19F NMR spectroscopy at 470 MHz (11.7 Tesla) has been used to directly measure the levels of 5-fluorouracil (FU) and its fluorine-containing catabolites in plasma and urine of colon cancer patients after i.v. infusion (10 min) of 60-230 mumol (8-30 mg) FU/kg, either with or without pretreatment with methotrexate (5.1-12.5 mg/kg). With a 1.5-ml sample the minimum metabolite concentration that can be quantified is approximately 15 +/- 5 microM within 30 min and 3 +/- 1 microM within 12 h of data acquisition. The first and second catabolites of FU, dihydrofluorouracil and alpha-fluoro-beta-ureidopropanoic acid, exhibit steady-state behavior with dose-dependent plasma concentrations of 5-40 microM for approximately 10-90 min after infusion (12 patients, 16 treatments). The final catabolite alpha-fluoro-beta-alanine (FBAL) was detected in plasma after 5-15 min, and the rate at which its concentration increased was independent of FU dose, while the maximum concentration reached at about the time FU disappeared (FU less than 5 microM in 1-2 h) was dose-dependent. The area under the time curve for FU in plasma increased more than linearly with dose. Several patients showed elevated levels of free fluoride anion (F-) in plasma (63 samples: median, 5 microM; maximum, 33 microM). In urine all of the above catabolites and F- could be observed. In samples with pH greater than or equal to 7.3 (methotrexate patients, due to bicarbonate infusion) N-carboxy-FBAL was also found in significant amounts. Urinary excretion of FU and catabolites amounted to 2.6-30% of the dose within 2 h (14 patients, 18 treatments) and 60-66% within 24 h (three patients). The ratio FU/creatinine in 2-h urine increased more than linearly with FU dose. Urinary fluoride concentration reached a maximum during the first day after FU infusion and returned to normal background levels after 2-3 days (four patients). The pattern of FU catabolites observed in plasma or urine did not differ significantly between responders and nonresponders to therapy or between patients with FU monotherapy and patients with methotrexate pretreatment. Cytotoxic FU anabolites, i.e., nucleotides, were not detected in plasma or urine (i.e., are less than 3 microM). Their detection in tumor tissue will be required for an assessment of individual responsiveness to FU. Possible toxic metabolic products derivable from FBAL, e.g., 2-fluoroacetate or 2-fluorocitrate, were not detected (i.e., are less than 3 microM) in plasma or urine.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed1988

Chain-fluorinated polyamines as tumour markers. III. Determination of geminal difluoropolyamines and their precursor 2,2-difluoroputrescine in normal tissues and experimental tumours by in vitro and in vivo 19F NMR spectroscopy.

Treatment of laboratory animals bearing transplantable tumours with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, followed by 2,2-difluoroputrescine allows the partial replacement of the endogenous polyamines spermidine and spermine by their fluorine-containing analogs 6,6-difluorospermidine and 6,6-difluorospermine. Quantitative determination of the chain-fluorinated polyamines in excised tumor tissue by in vitro 19F NMR spectroscopy at 11.7 T gave results similar to those obtained by extraction and HPLC analysis. The NMR methods allowed the observation of several as yet unidentified geminal difluoropolyamine metabolites with a detection threshold as low as 1 nmole/g. Using a surface coil at 2.4 T, it was possible to detect in 10-20 min fluorinated polyamines in tumour, liver and bladder (urine) of mice in vivo at concentrations of the order of 50-100 nmol/g, i.e., when 2-5% of the natural polyamines have been replaced by their fluoro analogs. Since 6,6-difluorospermidine and 6,6-difluorospermine preferentially accumulate in tumours and specific tissues with enhanced polyamine metabolism, it is expected that these compounds may serve as probes for the application of 19F NMR methods for tumour diagnosis and therapy control. Since significant quantities of additional fluorine-containing polyamine metabolites were detected in some tissues, 19F NMR spectroscopy will contribute to our knowledge of polyamine metabolism in normal and tumour tissue.

Animals↗
PubMed1988

Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.

Animals↗
PubMed1988

Quantitative aspects of accelerated nuclear polyploidization and tumour formation in dieldrin treated CF-1 mouse liver.

Nuclear polyploidization in the livers of CF-1 mice, exposed to dieldrin (0, 1, 5 and 10 ppm in the diet), was studied up to the median time of liver tumour development (ranging from 15 to 27 months) in the respective treatment groups. In untreated controls nuclear polyploidization is characterized by a linear increase of octaploid nuclei with age. Approximately 4 months before tumour development a reduction in the tetraploid to diploid ratio is observed. Dieldrin treatment was found to enhance nuclear polyploidization in the initial phases of treatment, as expressed by a dose-dependent increase in octaploid nuclei. In 'steady-state' situations all age dependent changes in the level of polyploidization found in controls were also found in dieldrin treated mice. However, these changes occurred at an increasingly earlier age with higher dieldrin treatment levels. The decrease in the tetraploid:diploid ratio always takes place a few months before tumour development. This change in the ploidy level may thus be related to the subsequent liver tumour formation. The liver tumours themselves appear to originate from a diploid stem line, and were found to increase their degree of polyploidization during growth, eventually developing aneuploid nuclei. A comparison of nuclear polyploidization and liver tumour formation in CF-1 mouse liver for the given dietary dieldrin concentrations showed that liver tumour formation was associated with a constant level of polyploidization. Since polyploidization is an age-dependent process, these findings suggest that liver tumour formation is imminent at a constant biological age and that dieldrin may advance the biological age of CF-1 mouse liver.

Administration, Oral↗
PubMed1987

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa.

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.

Adenine Nucleotides↗
PubMed1987

Development of cytochrome P-450-altered preneoplastic and neoplastic lesions during nitrosamine-induced hepatocarcinogenesis in the rat.

The expression of four cytochrome (cyt.) P-450 isoenzymes has been studied in preneoplastic and neoplastic lesions during the course of nitrosamine-induced hepatocarcinogenesis in the female Wistar rat. Following exposure to diethylnitrosamine (50 or 100 ppm in the drinking water) for 10 days, animals were taken sequentially, and the livers were analyzed for the evolution of adenosine triphosphatase deficient focal lesions. These lesions were subdivided into different phenotypes with regard to their cyt. P-450 isoenzyme expression using serial frozen sections. Our results demonstrate that about 40% of the adenosine triphosphatase-deficient lesions show concomitant alterations in their cyt. P-450 isoenzyme contents. Of these lesions, islets which are characterized by decreased levels of at least three cyt. P-450 isoenzymes show a dramatic increase in their volumetric fraction of liver tissue with progression of time. Although only very few lesions express this phenotype, the contribution to the volumetric fraction of islet tissue raises from about 2% at 10 weeks to about 60% at 35 weeks after cessation of diethylnitrosamine treatment. By contrast, lesions which express less than two alterations in cyt. P-450 isoenzyme levels develop relatively slowly. Similar results were obtained when animals were exposed continuously to diethylnitrosamine for a period of up to 8 weeks. Following treatment of islet-bearing animals with phenobarbital, an induction of cyt. P-450 isoenzymes and NADPH-cyt. P-450-reductase was observed within preneoplastic and neoplastic lesions. This induction was most pronounced in large, expansively growing nodules, a type of lesion which displayed decreased levels of these enzymes in livers of animals not treated with phenobarbital. The elevation of the cyt. P-450 isoenzymes disappeared within 2 to 3 weeks after cessation of inducer treatment. Our results indicate that a high proportion of rapidly growing lesions has assumed a constitutive deficiency in cyt. P-450 isoenzyme expression during nitrosamine-induced hepatocarcinogenesis. This deficiency, however, is not an irreversible quality, since individual cyt. P-450 isoenzymes can be markedly induced by treatment with an enzyme inducer like phenobarbital. Thus, the observed decrease in cyt. P-450 expression during development of malignancy does not result from alterations in the cyt. P-450 encoding structural genes but may rather be related to abnormalities in the function of regulatory systems of a higher order which may play a central role in the maintenance of cell homeostasis.

Animals↗
PubMed1987

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria.

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

Adenine Nucleotides↗