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J Wilhelm

Publications and source records attributed to J Wilhelm.

At least 37 records · Page 2Linked to original sources
PubMed2001

Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes.

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.

DNA-Directed DNA Polymerase↗
PubMed2001

The effects of hyperoxia, hypoxia, and ischemia/reperfusion on the activity of cytochrome oxidase from the rat retina.

Cytochrome oxidase activity from the retina can be enhanced or depressed by free radical-mediated reactions both in positive and negative aspect. The greatest effect was exerted by ischemia/reperfusion, which significantly increased the fluorescent products of lipid peroxidation (358 %, P < 0.01) and inhibited the enzyme activity (14%, P < 0.001). After hyperoxia the fluorescent products slightly increased (192%, P < 0.05) as well as the enzyme activity (133 %, P < 0.05). Hypoxia had no effect on any of these parameters. Specific changes in the composition of fluorophores after ischemia/reperfusion were revealed in the fluorescence spectra. The fact that increased lipid peroxidation after hyperoxia and after ischemia/reperfusion does not produce the same effect upon cytochrome oxidase activity might be explained by changes in the kinetic behavior of cytochrome oxidase. In the control enzyme preparation, two binding sites for cytochrome c were observed. One was of the low-affinity (Km = 60 microM) and the other of the high-affinity (Km = 1.12 microM). After in vitro-initiated lipid peroxidation, the low-affinity binding site was lost and the activity measured under "optimum" conditions at a single cytochrome concentration was higher than in the controls. This implies that oxidative damage to cytochrome oxidase in vivo can be site-specific and its extent should be estimated by performing detailed kinetic analysis as otherwise the results might be misleading.

Animals↗
PubMed2000

[Role of nonenzymatic glycation and oxidative stress on the development of complicated diabetic cataracts].

Eye lens cataract can develop by various mechanisms the details of which are not completely known. Increased level of glucose in patients with diabetes mellitus represents one of the factors accelerating cataract development. As the lens does not depend on insulin, cataract formation is induced by hyperglycaemia both in IDDM and NIDDM patients. Glucose attacks free aminogroups of proteins and glycation products are formed by multistep non-enzymatic reactions. We can discriminate early and late products. The latter are often called as advanced glycation endproducts (AGE). They can accumulate inside the lens and interfere with its optical properties. With regard to the fact that glycation reactions are accompanied by autooxidation reactions, the overall process is usually referred to as glycoxidation. Free radical oxidations of membrane lipids give similar products as do glycation reactions, and there is synergy between these two pathways. Development of eye cataract is accompanied by a decrease in lens antioxidant capacity. A new class of glycation inhibitors has been observed recently. They are called Amadorins and they have therapeutic potential in the cataract treatment.

Cataract↗
PubMed2000

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2000

A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension.

Chronic sojourn in hypoxic environment results in the structural remodeling of peripheral pulmonary arteries and pulmonary hypertension. We hypothesize that the pathogenesis of changes in pulmonary vascular structure is related to the increase of radical production induced by lung tissue hypoxia. Hypoxia primes alveolar macrophages to produce more hydrogen peroxide. Furthermore, the increased release of oxygen radicals by other hypoxic lung cells cannot be excluded. Several recent reports demonstrate the oxidant damage of lungs exposed to chronic hypoxia. The production of nitric oxide is high in animals with hypoxic pulmonary hypertension and the serum concentration of nitrotyrosine (radical product of nitric oxide and superoxide interaction) is also increased in chronically hypoxic rats. Antioxidants were shown to be effective in the prevention of hypoxia induced pulmonary hypertension. We suppose that the mechanism by which the radicals stimulate of the vascular remodeling is due to their effect on the metabolism of vascular wall matrix proteins. Non-enzymatic protein alterations and/or activation of collagenolytic matrix metalloproteinases may also participate. The presence of low-molecular weight cleavage products of matrix proteins stimulates the mesenchymal proliferation in the wall of distal pulmonary arteries. Thickened and less compliant peripheral pulmonary vasculature is then more resistant to the blood flow and the hypoxic pulmonary hypertension is developed.

Animals↗
PubMed1999

NMR solution structure of the catalytic fragment of human fibroblast collagenase complexed with a sulfonamide derivative of a hydroxamic acid compound.

The solution structure of the catalytic fragment of human fibroblast collagenase (MMP-1) complexed with a sulfonamide derivative of a hydroxamic acid compound (CGS-27023A) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. The solution structure of the complex was calculated by means of hybrid distance geometry-simulated annealing using a combination of experimental NMR restraints obtained from the previous refinement of the inhibitor-free MMP-1 (1) and recent restraints for the MMP-1:CGS-27023A complex. The hydroxamic acid moiety of CGS-27023A was found to chelate to the "right" of the catalytic zinc where the p-methoxyphenyl sits in the S1' active-site pocket, the isopropyl group is in contact with H83 and N80, and the pyridine ring is solvent exposed. The sulfonyl oxygens are in hydrogen-bonding distance to the backbone NHs of L81 and A82. This is similar to the conformation determined by NMR of the inhibitor bound to stromelysin (2, 3). A total of 48 distance restraints were observed between MMP-1 and CGS-27023A from 3D 13C-edited/12C-filtered NOESY and 3D 15N-edited NOESY experiments. An additional 18 intramolecular restraints were observed for CGS-27023A from a 2D 12C-filtered NOESY experiment. A minimal set of NMR experiments in combination with the free MMP-1 assignments were used to assign the MMP-1 (1)H, 13C, and 15N resonances in the MMP-1:CGS-27023A complex. The assignments of CGS-27023A in the complex were obtained from 2D 12C-filtered NOESY and 2D 12C-filtered TOCSY experiments.

Catalysis↗
PubMed1999

Hypoxia induces free radical damage to rat erythrocytes and spleen: analysis of the fluorescent end-products of lipid peroxidation.

Several studies have shown that hypoxia induces alterations in the lipid membranes of many cell types. The mechanism of these changes might consist in membrane lipid peroxidation. Lipid peroxidation in erythrocytes and spleen is easily detected by measurement of the concentration of fluorescent end-products. Exposure of rats to hypoxia for various time periods induced formation of lipophilic fluorescent products both in erythrocytes and spleen. A new kind of fluorophore was found in chloroform extracts from erythrocytes with excitation maximum at 270 nm and emission maximum at 310 nm. Additionally, two minor fluorophores were observed, emitting at 360 nm and in the region of 415-440 nm. Only one type of fluorophore was detected in spleen, emitting at 445 nm after excitation at 315 nm. The concentration of fluorophores was dependent on the time of hypoxic exposure both in erythrocytes and spleen. In erythrocytes there was a decrease of the predominant fluorophore after 3 hours (54%, P < 0.05) and 21 days (54%, P < 0.05) of hypoxia in relation to normoxic controls, accompanied by changes in spectral patterns of tridimensional fluorescence spectra. There was also a significant increase in the concentration of fluorophore in spleen (to 164%, P < 0.05, after 3 h, and to 240%, P < 0.05, after 21 days). The fluorophores, both in erythrocytes and spleen, were resolved into several distinct fractions with HPLC. The presented results support the hypothesis of hypoxia-induced lipid peroxidation and create a basis for further characterization of the fluorescent products.

Animals↗
PubMed1999

Influence of macrophages and macrophage-modified collagen I on the adhesion and proliferation of vascular smooth muscle cells in culture.

The adhesion, proliferation and morphology of rat vascular smooth muscle cells (VSMC) in cocultures with macrophages or in cultures on type I collagen modified by activated macrophages were evaluated. In the first set of experiments, rat alveolar macrophages were added to 24-hour-old VSMC cultures. Between days 2 and 5 after VSMC seeding, the population densities and doubling times of cells were similar in both VSMC-macrophage and pure VSMC cultures. However, from day 5, the cocultures proliferated about two times more rapidly and on day 7, they reached higher cell population density by 40%. The pure macrophage cultures did not proliferate. In the second set of experiments, rat alveolar macrophages were activated by non-toxic TiO2 dust to produce reactive oxygen species and incubated for 120 min with collagen I. The collagen was then adsorbed on plastic culture dishes and seeded with VSMC. The collagen exposed for 10 min only, the unmodified collagen and pure culture dishes were used as control growth supports. On all four tested substrates, the number of initially adhered cells was similar, but on the collagen modified for 120 min, the cells were less spread. Moreover, on day 2 to 3 after seeding, some cells on this collagen became vacuolated and detached spontaneously from the growth support. The remaining VSMC, however, rapidly proliferated, so that on day 9, the cell population density on 120-min-modified collagen was similar as on both control collagens and significantly higher compared to that on uncoated dishes. Our results suggest that 1. The delayed growth-stimulating effect of macrophages on VSMC-macrophage mixed population is probably due to autocrine production of mitogens by both cell types rather than due to an acute effect of short-living oxygen radicals released from macrophages immediately after adding to VSMC cultures. 2. The effect of collagen I exposed to activated macrophages for 120 min is slightly cytotoxic, which could, however, stimulate a release of mitogens from damaged as well as surviving VSMC.

Animals↗
PubMed1999

Hydrogen peroxide in the breath of rats: the effects of hypoxia and paraquat.

The hypothesis that oxidative stress can be induced by hypoxia was tested by measuring the concentration of hydrogen peroxide by a luminometric technique in the breath samples of rats exposed to hypoxia and paraquat. The group of animals (n=15) exposed to normobaric hypoxia (10% O2) for three days had an increased amount of H2O2 (200%, P<0.001) in their breath in comparison to control animals. After 7 days of recovery in air, the exposed animals still produced significantly increased levels of H2O2 (152%, P<0.001). Paraquat administration was used as a positive control, since it is a redox cycling compound producing free radicals. In the animals treated with a toxic dose of paraquat, the peak H2O2 production was observed 5 h after i.p. injection (156%, P<0.02). Within the next 2 h it decreased to the control level and stayed constant for 48 h, when the animals began to die. It is suggested that H2O2, observed in the breath samples, is a product of a metabolic pathway that could itself be sensitive to oxidative damage.

Animals↗
PubMed1998

Expressed sequences as candidates for a novel tumor suppressor gene at band 13q14 in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.

Deletions affecting the interval between the RB1 gene and marker D13S25 at band 13q14 are the most frequent genetic abnormalities of B-cell chronic lymphocytic leukemia (B-CLL) and indicate the presence of a novel tumor suppressor gene in this region. In the current study, a high resolution physical map of fragments spanning one megabasepair (Mb) of genomic DNA at the critical 13q14 segment was constructed. To define the minimal region of loss within the RB1 and D13S25 interval, we screened 322 B-CLLs for deletions at either of the two loci. Thirty mantle cell lymphomas (MCLs) were included in the analysis because we observed a 13q14 deletion pattern similar to B-CLL in this disease. The incidence of 13q14 deletions was 51% in B-CLL and 70% in MCL, respectively. No frequent loss of the BRCA2 gene at band 13q12 was found. Detailed deletion mapping at band 13q14 with probes from the RB1-D13S25 interval lead to the identification of a critical deletion region 400 kb in size. Within this region two segments were most frequently affected, one at D13S272 120 kb in size and another 240 kb distal of D13S272 80 kb in size. From these two segments expressed sequences were identified as candidates for the putative 13q14 tumor suppressor gene involved in the pathogenesis of B-CLL and MCL.

BRCA2 Protein↗
PubMed1998

High-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase determined by multidimensional NMR.

The high-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase (MMP-1), a protein of 18.7 kDa, which is a member of the matrix metalloproteinase family, has been determined using three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 3333 experimental NMR restraints, consisting of 2409 approximate interproton distance restraints, 84 distance restraints for 42 backbone hydrogen bonds, 426 torsion angle restraints, 125 3JNH alpha restraints, 153 C alpha restraints, and 136 C beta restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures for residues 7-137 and 145-163 is 0.42 +/- 0.04 A for the backbone atoms, 0.80 +/- 0.04 A for all atoms, and 0.50 +/- 0.03 A for all atoms excluding disordered side chains. The overall structure of MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and anti-parallel arrangement and three alpha-helices. A best-fit superposition of the NMR structure of inhibitor-free MMP-1 with the 1.56 A resolution X-ray structure by Spurlino et al. [Spurlino, J. C., Smallwood, A. M., Carlton, D. D., Banks, T. M., Vavra, K. J., Johnson, J. S., Cook, E. R., Falvo, J., and Wahl, R. C., et al. (1994) Proteins: Struct., Funct., Genet. 19, 98-109] complexed with a hydroxamate inhibitor yields a backbone atomic rms difference of 1.22 A. The majority of differences between the NMR and X-ray structure occur in the vicinity of the active site for MMP-1. This includes an increase in mobility for residues 138-144 and a displacement for the Ca(2+)-loop (residues 74-80). Distinct differences were observed for side-chain torsion angles, in particular, the chi 1 for N80 is -60 degrees in the NMR structure compared to 180 degrees in the X-ray. This results in the side chain of N80 occupying and partially blocking access to the active site of MMP-1.

Binding Sites↗
PubMed1997

Oxidized collagen stimulates proliferation of vascular smooth muscle cells.

We hypothesize that the vascular smooth muscle proliferation after lung injury results from oxidative damage to the matrix proteins in the walls of pulmonary blood vessels. The smooth muscle cells (SMC) isolated from rat aorta were cultured on the surface coated with oxidized and nonoxidized (control) collagen of type I. Oxidation of collagen was induced by UV irradiation and characterized by fluorescence tridimensional spectral arrays and by gel electrophoresis. From day 1 to 6 of the experiment, SMC proliferated more rapidly on the oxidized collagen than on the control surface. At high SMC population densities (day 9 of experiment) the difference disappeared. After 10 min of trypsinization the cells growing on oxidized collagen rounded and detached completely from the growth surface. The control cells on nonoxidized collagen detached only after 30 min of trypsinization. We conclude that oxidation of collagen of vascular wall matrix may participate in stimulation of SMC proliferation after oxidant tissue injury.

Animals↗
PubMed1997

Production of hydrogen peroxide by peritoneal macrophages from rats exposed to subacute and chronic hypoxia.

Hydrogen peroxide production was measured in non-elicited rat peritoneal macrophages using luminol-dependent chemiluminescence (LDCL). Isolated cells were activated by a chemotactic peptide (FMLP) or by a phorbol ester (PMA) or by the combination of both. A hundred-fold higher LDCL intensity was achieved with PMA relative to FMLP. However, when FMLP was added subsequently to PMA it produced approximately the same response as did PMA. These measurements were carried out with cells isolated from controls and from animals exposed to normobaric hypoxia (10% O2) for 3 hours, 3 days, or 21 days. Hypoxia had a dual effect. Acutely (within 3 hours) it attenuated the production of hydrogen peroxide triggered by PMA, whilst during longer exposure (3 or 21 days) it increased the response induced by FMLP. Hypoxia can thus modulate the capacity of respiratory burst in peritoneal macrophages.

Animals↗
PubMed1996

Production of hydrogen peroxide by alveolar macrophages from rats exposed to subacute and chronic hypoxia.

We have studied in vitro alveolar macrophages (AMs) obtained by tracheobronchial lavage from rats exposed to subacute (3 hours and 3 days) and chronic (3 weeks) hypoxia (FiO2 = 0.1) and from rats recovering from chronic hypoxia. Hydrogen peroxide production by AMs was measured by luminol-dependent chemiluminescence after AMs adhered to the walls of the measuring cuvette, after stimulation with phorbol-myristate-acetate (PMA), and when N-formyl-methionyl-leucyl-phenylanine (FMLP) was added subsequently to the cells which had been previously stimulated by adherence or PMA. H2O2 production after cell adherence and adherence combined with FMLP stimulation did not differ between the groups. The increase of H2O2 production after adding PMA, and FMLP in addition to PMA was significantly higher in AMs from rats exposed to hypoxia for 3 days than in the controls. Other experimental groups did not differ from their controls. It is concluded that 3 days' hypoxia primes AMs for enhanced production of H2O2 upon stimulation. The mechanism is probably at the level of synthesis of proteins involved in H2O2 production, or the shift to a more reactive phenotype of alveolar macrophages subpopulations.

Animals↗
PubMed1995

[Reactive oxygen species and homeostasis].

Oxygen represents an important reactant which plays both positive and negative role in the metabolism. The deleterious effects are caused by the reactivity of substances originating in the partial reductions of their molecules. Therefore in the course of evolution many antioxidant systems have been developed, both enzymic and non-enzymic. The present review describes individual reactive oxygen species and the way of their detoxication in the organism. An important feature of antioxidant defence is its compartmentalization and cooperation between individual antioxidant systems which are described also with respect to their genetic encoding. For comparison properties of some artificial widely-used antioxidants are shown and their biological effects are discussed.

Animals↗
PubMed1995

Role of ion fluxes in hydrogen peroxide pulmonary vasoconstriction.

Hydrogen peroxide injected into the inflow cannula of isolated ventilated rat lungs produced a dose-dependent vasoconstriction in the range 0.25-10 mM, with maximum response between 2-5 mM. The effects of H2O2 can be influenced by ionophores or specific inhibitors of ionic channels or pumps. A key role is played by sodium ions which govern the subsequent inflow or outflow of calcium, an ion mediating the vasoconstriction. A physiological role for H2O2 generated by NADPH oxidase is postulated.

Animals↗
PubMed1995

Production of hydrogen peroxide by alveolar macrophages. Effect of barbiturates.

Production of hydrogen peroxide by rat lung alveolar macrophages represents one of the key events in the inflammatory process. For the interpretation of the in vitro measurements it is important to control all possible interfering influences. The present work documents that the type of anaesthesia might critically influence the observed results. H2O2 production was measured in isolated rat alveolar macrophages by luminol chemiluminescence catalyzed by horseradish peroxidase. Three different mechanisms of H2O2 production were observed after stimulation of cells with a chemotactic peptide (FMLP), phorbol ester (PMA), and during cell adherence. All these activities were influenced independently by the treatment with barbiturates, which both stimulated or inhibited the H2O2 production, depending on the barbiturate concentration. As the effective barbiturate concentrations were found to be within the range used for the anaesthesia of experimental animals, the presented results imply that barbiturates are not suitable for experiments in which the production of reactive oxygen species by phagocytes is measured, and that other anaesthetics should be tested.

Animals↗