Biomedical subjects

G Fritz

Publications and source records attributed to G Fritz.

At least 37 records · Page 2Linked to original sources

PubMed2001

Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6.

Cytochrome c3, a small (14-kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high-molecular-mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane-associated dissimilatory sulfite reductase. The EPR spectra show features of four different low-spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X-ray diffraction data were collected to below 2 A resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.

Amino Acid Sequence↗

PubMed2001

Lipoprotein lipase mediates the uptake of glycated LDL in fibroblasts, endothelial cells, and macrophages.

The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R-related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered "proatherogenic." Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R-deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.

Acetylation↗

PubMed2000

Ras-related GTPase Rhob represses NF-kappaB signaling.

rhoB encoding a Ras-related GTPase is immediate-early inducible by genotoxic treatments, indicating that it is part of the cellular stress response. Here, we investigated the influence of RhoB on signal pathways that are rapidly evoked by genotoxic compounds. The data obtained show that wild-type RhoB neither affects activation of mitogen-activated protein kinases nor AP-1-dependent gene expression. However, RhoB inhibited both basal and genotoxic agent-stimulated activity of the transcription factor nuclear factor kappaB (NF-kappaB). Thus, RhoB attenuated alkylation-induced increase in the DNA binding activity of NF-kappaB and abrogated NF-kappaB-driven gene expression. Furthermore, RhoB inhibited decrease in the cellular amount of IkappaBalpha after genotoxic stress as well as after tumor necrosis factor alpha and 12-O-tetradecanoylphorbol acetate treatment. This indicates that RhoB represses NF-kappaB activation by inhibiting dissociation and subsequent degradation of IkappaBalpha. On the basis of the data, we suggest that RhoB is a novel negative regulator of NF-kappaB signaling.

3T3 Cells↗

PubMed2000

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions. Therefore, this method was used to subsequently compare the effectiveness of MPO inhibitors that block production of HOCl with compounds that act as HOCl traps. The efficiency of MPO inhibitors to prevent LDL damage increased in the series benzohydroxamic acid < salicylhydroxamic acid < 3-amino-1,2,4-triazole < sodium azide < potassium cyanide < p-hydroxy-benzoic acid hydrazide, while for the HOCl traps the protective efficiency increased in the series glycine < taurine < methionine. We conclude that HOCl traps may have high potential therapeutic impact in vivo due to their low toxicity, although high concentrations of them would have to reach sites of inflammation. In contrast, only low concentrations of a specific MPO inhibitor would be required to irreversibly inhibit the enzyme.

Arteriosclerosis↗

PubMed2000

Adenylylsulfate reductases from archaea and bacteria are 1:1 alphabeta-heterodimeric iron-sulfur flavoenzymes--high similarity of molecular properties emphasizes their central role in sulfur metabolism.

Highly active adenylylsulfate (APS) reductase was isolated under N(2)/H(2) from sulfate-reducing and sulfide-oxidizing bacteria and archaea. It was a 1:1 alphabeta-heterodimer of molecular mass approximately 95 kDa, and two subunits (alpha approximately 75, beta approximately 20 kDa). The specific activity was 11-14 micromol (min mg)(-1); cofactor analysis revealed 0.96+/-0.05 FAD, 7.5+/-0.1 Fe and 7.9+/-0.25 S(2-). The photochemically reduced enzyme had a multiline EPR spectrum resulting from two interacting [4Fe-4S] centers. The properties of the different APS reductases were remarkably similar, although the enzyme is involved in different metabolic pathways and was isolated from phylogenetically far separated organisms. A structural model is proposed, with FAD bound to the alpha-subunit, and two [4Fe-4S] centers located in close proximity on the beta-subunit.

Adenosine Monophosphate↗

PubMed2000

Ras-related GTPase RhoB forces alkylation-induced apoptotic cell death.

rhoB encoding a Ras-related GTPase is immediate-early inducible by genotoxic treatments. To address the question of the physiological role of RhoB in cellular defense, cells stably overexpressing wild-type RhoB protein were generated. Overexpression of RhoB renders cells hypersensitive to the killing effect of alkylating agents including antineoplastic drugs but not to UV-light and doxorubicin. As compared to control cells, RhoB overexpressing cells revealed an increase in the frequency of alkylation-induced apoptotic cell death. This indicates that RhoB is involved in modulating apoptotic signaling. Furthermore, overexpression of RhoB resulted in a prolonged transient block to DNA replication upon MMS treatment. UV-induced replication blockage was not affected by RhoB. Based on the data we suggest RhoB to be a novel regulatory factor which takes influence on the level of cytotoxicity of DNA damaging drugs and forces cells to alkylation-induced apoptosis. The data indicate that this might be due to RhoB mediated delay in cell cycle progression upon alkylation treatment.

3T3 Cells↗

PubMed2000

Human APE/Ref-1 protein.

Human apurinic (apyrimidinic) endonuclease/redox-factor 1 (APE/Ref-1) is an ubiquitously expressed multifunctional protein of 36.5 kDa which is encoded by a approximately 3 kb gene localized on chromosome 14. APE/Ref-1 is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. The repair function of the protein is located on its C-terminal region. Activation of transcription factors, which occurs via a redox-based mechanism, pertains to a approximately 6 kDa N-terminal fragment with Cys-65 being of essential importance. APE/Ref-1 is part of the cellular response to oxidative stress and protects cells from the genotoxic and cytotoxic effect of oxidizing agents. Thus, specific downmodulation of APE/Ref-1 activity in tumors might be considered as a novel therapeutic approach in tumor therapy.

Carbon-Oxygen Lyases↗

PubMed2000

Helicobacter pylori prevalence in Leipzig's 1998 school entries: methodology and first results.

The non-invasive, stable-isotope-aided Helicobacter pylori (H. pylori) tests--breath and equivalent urine tests--were offered on a voluntary basis as part of the mandatory school entry medical examination to the 1998 school entry cohort of the City of Leipzig (480,000 residents). Parents of participating subjects were asked to fill out a detailed epidemiologic questionnaire. The response rate was 94% (n = 2228 of 2369 school starters born in 1991/92). Parent-completed questionnaires were returned by 1890 (80%) children. The overall H. pylori positive prevalence was 7.2%. The prevalence among children with a test and a parent-completed questionnaire was 6.5%. Prevalences among subsequently tested family members of the positive tested children was 65, 60 and 39% for mothers, fathers and siblings respectively. Though studies have shown that the direct transmission of the bacterium (oral-oral and fecal-oral) is a dominant pathway of infection, the questionnaire analyses indicate associations between H. pylori colonisation and living as well as environmental conditions.

Breath Tests↗

PubMed2000

Crystallization and preliminary X-ray analysis of adenylylsulfate reductase from Archaeoglobus fulgidus.

A group of anaerobic microorganisms use sulfate as the terminal electron acceptor for energy conservation. The process of sulfate reduction involves several enzymatic steps. One of them is the conversion of adenylyl sulfate (adenosine-5'-phosphosulfate) to sulfite, catalyzed by adenylylsulfate reductase. This enzyme is composed of a FAD-containing alpha-subunit and a beta-subunit harbouring two [4Fe-4S] clusters. Adenylylsulfate reductase was isolated from Archaeoglobus fulgidus under anaerobic conditions and crystallized using the hanging-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals grew in space group P2(1)2(1)2(1), with unit-cell parameters a = 72.4, b = 113.2, c = 194.0 A. The asymmetric unit probably contains two alphabeta units. The crystals diffract beyond 2 A resolution and are suitable for X-ray structure analysis.

Archaeoglobus fulgidus↗

PubMed2000

Inhibition of protein isoprenylation impairs rho-regulated early cellular response to genotoxic stress.

Activation of c-Jun N-terminal kinases (JNKs) and nuclear factor-kappaB (NF-kappaB) are early cellular responses to genotoxic stress involved in the regulation of gene expression. Pretreatment of cells with the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin blocked stimulation of JNK1 activity by UV irradiation and by treatment with the alkylating compound methyl methanesulfonate but did not affect activation of extracellular signal-regulated kinase 2 by UV light. Lovastatin also attenuated UV-induced degradation of the NF-kappaB inhibitor IkappaBalpha. The effects of lovastatin on UV-triggered stimulation of JNK1 as well as on IkappaBalpha degradation were reverted by cotreatment with geranylgeranylpyrophosphate but not with farnesylpyrophosphate. Both a geranylgeranyltransferase type I inhibitor and a farnesyltransferase inhibitor blocked JNK1 stimulation by UV irradiation without impairing signaling to NF-kappaB. This indicates that different types of isoprenylated proteins impair UV-induced signaling to JNK1 and NF-kappaB, respectively. Since lovastatin caused a rapid decrease in the level of membrane-bound Rho GTPases, we hypothesize that Rho signaling is inhibited by lovastatin. In line with this hypothesis, Rho-inactivating toxin B from Clostridium difficile abolished both JNK1 activation and IkappaBalpha degradation evoked by UV irradiation. In summary, lovastatin-mediated inhibition of protein isoprenylation abrogates cellular stress responses involving JNK- and NF-kappaB-regulated pathways, which seems to be caused by inactivation of Rho GTPases.

Animals↗

PubMed1999

Rho GTPases are over-expressed in human tumors.

Small GTPases of the Rho family are involved in the regulation of a variety of cellular processes, such as the organization of the microfilamental network, cell-cell contact and malignant transformation. To address the question of whether Rho proteins are involved in carcinogenesis in man, we compared their expression in tumors from colon, breast and lung with that of the corresponding normal tissue originating from the same patient. As shown by Rho-specific 32P-ADP-ribosylation, as well as Western-blot analysis, the amount of RhoA protein was largely increased in all 3 types of tumors tested. The most dramatic differences in the expression of Rho GTPases were observed in breast tissue. All breast tumors analyzed showed high levels of RhoA, Rac and Cdc42 proteins, whereas in the corresponding normal tissue these Rho proteins were hardly or not detectable. Progression of breast tumors from WHO grade I to grade III was accompanied by a significant average increase in RhoA protein. Overall, increase in the amount of Rho GTPases, in particular RhoA, appears to be a frequent event in different types of human tumors. This supports the view that Rho GTPases are involved in human carcinogenesis.

Adenosine Diphosphate Ribose↗

PubMed1999

Characterization of Enzymatically Induced Aggregation of Casein Micelles in Natural Concentration by in Situ Static Light Scattering and Ultra Low Shear Viscosimetry.

The aggregation of casein micelles in undiluted skim milk after the addition of chymosin was studied by static light scattering and ultra low shear viscometry. The static light scattering measurements were made with two different sample thicknesses, 72 and 16 µm. The scattering data were analyzed by indirect Fourier transformation and by the polydispersity inversion technique which led to pair distance distribution functions and size distribution function, respectively. The minimum scattering angle was 1 degrees, which allows for the determination of particle sizes up to a maximum diameter of 12 µm. The fractal dimension determined from double logarithmic plots of intensity versus scattering vector resulted in values between 1.9 and 2.0. The influence of multiple scattering was determined by comparison of the measurements with the different sample thicknesses. The measurements show no significant influence of multiple scattering when the transmission is above 0.85. Due to the very complex and porous structure of the casein aggregates the Rayleigh-Debye-Gans scattering theory has been used in the data analysis. Measurements with a new instrument using ultra low shear showed good agreement with theory. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Phosphorylation of the DNA repair protein APE/REF-1 by CKII affects redox regulation of AP-1.

The DNA repair protein apurinic endonuclease (APE/Ref-1) exerts several physiological functions such as cleavage of apurinic/apyrimidinic sites and redox regulation of the transcription factor AP-1, whose activation is part of the cellular response to DNA damaging treatments. Here we demonstrate that APE/Ref-1 is phosphorylated by casein kinase II (CKII). This was shown for both the recombinant APE/Ref-1 protein (Km=0.55 mM) and for APE/Ref-1 expressed in COS cells. Phosphorylation of APE/Ref-1 did not alter the repair activity of the enzyme, whereas it stimulated its redox capability towards AP-1, thus promoting DNA binding activity of AP-1. Inhibition of CKII mediated phosphorylation of APE/Ref-1 blocked mutagen-stimulated increase in AP-1 binding. It also abrogated the induction of c-Jun protein and rendered cells more sensitive to induced DNA damage. Thus, phosphorylation of APE/Ref-1 appears to be involved in regulating the different physiological activities of the enzyme. CKII mediated phosphorylation of APE/Ref-1 and concomitant increase in AP-1 binding activity appears to be a novel mechanism of cellular stress response, forcing transcription of AP-1 target gene(s) the product(s) of which may exert protective function.

Animals↗

PubMed1999

Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-iun expression.

Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression.

3T3 Cells↗

PubMed1998

Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3: spectroscopic investigations of its transition metal ion complexes, and three-dimensional structural model.

A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.

Amino Acid Sequence↗

PubMed1998

Apurinic endonuclease (Ref-1) is induced in mammalian cells by oxidative stress and involved in clastogenic adaptation.

Apurinic endonuclease (APE; also known as Ref-1 protein) is a key enzyme in base excision repair, cleaving apurinic sites that arise spontaneously because of the activity of DNA glycosylases. To address the question of whether APE can be modulated by genotoxic stress affecting cellular protection, we analyzed the expression of APE in Chinese hamster ovary (CHO) cells after treatment with various genotoxic agents. We show that treatment of CHO cells with hydrogen peroxide (H2O2) or sodium hypochlorite (NaOCl) increases the levels of APE mRNA and protein. APE induction was observed 3-9 h after treatment and was accompanied by an increase in APE activity. We also show that the cloned human APE promoter transfected into CHO cells is stimulated by the oxidants, indicating transcriptional activation of the APE gene. When cells were pretreated with NaOCl, inducing APE, and then challenged with H2O2, the clastogenic effect of the challenge dose was significantly reduced, suggesting clastogenic adaptation due to APE induction. To further prove the involvement of APE in adaptation against induced chromosomal breakage, we transfected human APE cDNA driven by an inducible promoter into CHO cells and observed that transient induction of APE reduced the clastogenic effect of H2O2. Overall, the data demonstrate that the APE gene can be activated by oxidative agents, resulting in a transient increase in APE repair activity, which reduces the clastogenic response of cells to an oxidative agent. The protection of cells from chromosomal aberrations seen after prior exposure to oxidants is attributed to an adaptive response to oxidative stress.

Animals↗

PubMed1998

Transgenic systems in studies on genotoxicity of alkylating agents: critical lesions, thresholds and defense mechanisms.

Transgenic systems, both cell lines and mice with gain or loss of function, are being used in order to modulate the expression of DNA repair proteins, thus allowing to assess their contribution to the defense against genotoxic mutagens and carcinogens. In this review, questions have been addressed concerning the use of transgenic systems in elucidating critical primary DNA lesions, their conversion into genotoxic endpoints, low-dose effects, and the relative contribution of individual cellular functions in defense. It has been shown that the repair protein alkyltransferase (MGMT) is decisive for protection against methylating and chloroethylating compounds. Protection pertains also to tumor formation, as revealed by the response of MGMT transgenic and knockout mice. Overexpression of genes involved in base excision repair (N-methylpurine-DNA glycosylase, apurinic endonuclease, DNA polymerase beta) is in most cases not beneficial in increasing the protection level, whereas their down-modulation or inactivation increases cellular sensitivity. This indicates that non-repaired base N-alkylation lesions and/or repair intermediates possess genotoxic potential. Modulation of mismatch repair and poly(ADP)ribosyl transferase has also been shown to affect the cellular response to alkylating agents. Furthermore, the role of Fos, Jun and p53 in cellular defense against alkylating mutagens is discussed.

Alkylating Agents↗

PubMed1998

Involvement of protein kinases in the induction of NO synthase II in human DLD-1 cells.

1. Protein phosphorylation is involved in the induction of nitric oxide synthase II (NOS II, iNOS) in several types of animal cells. Here we have investigated the possible involvement of major protein kinases in the induction of NOS II expression in human DLD-1 cells. 2. In DLD-1 cells, interferon--gamma alone induced a submaximal NOS II expression; a cytokine mixture consisting of interferon-gamma, tumour necrosis factor-alpha and interleukin-1beta produced maximal NOS II induction. 3. Activators of protein kinase A (forskolin, 8-dibutyryl-cyclic AMP), of protein kinase C (tetradecanoylphorbol-13-acetate), and of protein kinase G (8-bromo cyclic GMP) did not induce NOS II mRNA by themselves, nor did they alter NOS II mRNA induction in response to cytokines. 4. Inhibitors of protein kinase A (compound H89), of protein kinase C (bisindolylmaleimide, chelerythrine or staurosporine), of phosphatidylinositol 3-kinase (wortmannin), of p38 mitogen-activated protein kinase (compound SB 203580) and of extracellular signal-regulated kinase (compound PD 98059) also had no influence on basal or cytokine-induced NOS II mRNA expression. 5. Immunoprecipitation kinase assays showed no activation of extracellular signal-regulated kinase or p38 mitogen-activated protein kinase in cytokine-incubated DLD-1 cells. The c-Jun NH2-terminal kinase was activated by cytokines, but the most efficacious cytokine was tumour necrosis factor-alpha which did not induce NOS II by itself. 6. In contrast, the protein tyrosine kinase inhibitor tyrphostin B42 (a specific inhibitor of interferon-gamma-activated janus kinase 2) and the protein tyrosine kinase inhibitor tyrphostin A25 both reduced CM-induced NOS II mRNA expression in a concentration-dependent manner. 7. These results suggest that activation of NOS II expression in DLD-1 cells is independent of the activities of protein kinases A, C and G, phosphatidylinositol 3-kinase, extracellular signal regulated kinase and p38 mitogen-activated protein kinase, but seems to require protein tyrosine kinase activity, especially the interferon-gamma-activated janus kinase 2.

Calcium-Calmodulin-Dependent Protein Kinases↗