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Bernd Mayer

Publications and source records attributed to Bernd Mayer.

At least 19 recordsLinked to original sources
PubMed2007

Gene expression and biomarkers in renal transplant ischemia reperfusion injury.

The incidence of postischemic acute renal allograft failure (ARF) occurs in roughly 25% of cadaveric donor kidney recipients. This high rate remained virtually unchanged over the last decades despite modification in recipient management and modern immunosuppressive strategies. It has recently been shown that among other reasons, the systemic inflammation in the brain death cadaveric organ donor contributes to subsequent ARF in the recipient. This review focuses on the consequences of ischemia and reperfusion on the cellular level and offers potential solutions for the reduction of ARF. Genome-wide gene expression analysis together with sophisticated biostatistical analysis made it possible to identify several candidate gene products and proteins that may act as specific and sensitive biomarker for renal inflammation and ischemia. These markers may be very helpful in the clinical management of patients with a high a priori risk of subsequent ARF such as recipients of marginal donor kidneys. Ongoing clinical trials will evaluate whether immunosuppression of the cadaveric organ donor before organ harvest will have the potential to reduce inflammation in the transplant kidney and subsequently lead to a reduction in the rate of ARF.

Animals↗
PubMed2006

Nitric-oxide synthase: a cytochrome P450 family foster child.

Nitric-oxide synthase (NOS), the enzyme responsible for mammalian NO generation, is no cytochrome P450, but there are striking similarities between both enzymes. First and foremost, both are heme-thiolate proteins, employing the same prosthetic group to perform similar chemistry. Moreover, they share the same redox partner, a diflavoprotein reductase, which in the case of NOS is incorporated with the oxygenase in one polypeptide chain. There are, however, also conspicuous differences, such as the presence in NOS of the additional cofactor tetrahydrobiopterin, which is applied as an auxiliary electron donor to prevent decay of the oxyferrous complex to ferric heme and superoxide. In this review similarities and differences between NOS and cytochrome P450 are analyzed in an attempt to explain why NOS requires BH4 and why NO synthesis is not catalyzed by a member of the cytochrome P450 family.

Animals↗
PubMed2006

Inefficient spin trapping of superoxide in the presence of nitric-oxide: implications for studies on nitric-oxide synthase uncoupling.

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate L-arginine or the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H2O2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap. As expected, the Ca2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H2O2 in the absence of added L-arginine and BH4. Addition of BH4 (10 microM) did not significantly affect the rate of H2O2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H2O2. Although L-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H2O2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50-100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.

Animals↗
PubMed2006

Identification of a melanoma marker derived from melanoma-associated endogenous retroviruses.

We previously described the expression of melanoma-associated endogenous retrovirus (MERV) proteins and viral particles in human melanomas and metastases. The objective of the present study was to determine whether a humoral immune response to MERV proteins occurs in melanoma. Candidate B-cell epitopes on MERV proteins were predicted using bioinformatic screening. The reactivity of MERV peptides corresponding to the predicted epitopes with antibodies prevalent in sera of melanoma patients was analyzed. An immunodominant peptide located in the env protein of MERV was identified. Subsequent analyzes using 81 samples from stage I to stage IV melanoma patients and 95 sera from healthy subjects revealed statistically significant differences in seroprevalence of antibodies in melanoma sera samples when compared with reference samples from healthy subjects. The prevalence of anti-MERV antibodies in melanoma patient sera was confirmed by immunofluorescence on env-transfected cells. These data indicate the potential of this candidate peptide as target for diagnosis and immunotherapy.

Antibodies, Neoplasm↗
PubMed2006

Transforming omics data into context: bioinformatics on genomics and proteomics raw data.

Differential gene expression analysis and proteomics have exerted significant impact on the elucidation of concerted cellular processes, as simultaneous measurement of hundreds to thousands of individual objects on the level of RNA and protein ensembles became technically feasible. The availability of such data sets has promised a profound understanding of phenomena on an aggregate level, expressed as the phenotypic response (observables) of cells, e.g., in the presence of drugs, or characterization of cells and tissue displaying distinct patho-physiological states. However, the step of transforming these data into context, i.e., linking distinct expression or abundance patterns with phenotypic observables - and furthermore enabling a sound biological interpretation on the level of reaction networks and concerted pathways, is still a major shortcoming. This finding is certainly based on the enormous complexity embedded in cellular reaction networks, but a variety of computational approaches have been developed over the last few years to overcome these issues. This review provides an overview on computational procedures for analysis of genomic and proteomic data introducing a sequential analysis workflow: Explorative statistics for deriving a first, from the purely statistical viewpoint, relevant candidate gene/protein list, followed by co-regulation and network analysis to biologically expand this core list toward functional networks and pathways. The review on these procedures is complemented by example applications tailored at identification of disease-associated proteins. Optimization of computational procedures involved, in conjunction with the continuous increase in additional biological data, clearly has the potential of boosting our understanding of processes on a cell-wide level.

Animals↗
PubMed2006

Molecular signature of mice T lymphocytes following tolerance induction by allogeneic BMT and CD40-CD40L costimulation blockade.

Tolerance induction by mixed chimerism and costimulation blockade is a promising approach to avoid immunosuppression, but the molecular basis of tolerant T lymphocytes remains elusive. We investigated the genome-wide gene expression profile of murine T lymphocytes after tolerance induction by allogeneic bone marrow transplantation (BMT) and costimulatory blockade using the anti-CD40L antibody MR1. Molecular functions, biological processes, cellular locations, and coregulation of identified genes were determined. A total of 113 unique genes exhibited a significant differential expression between the lymphocytes of MR1-treated Tolerance (TOL) and untreated recipients Control (CTRL). The majority of genes upregulated in the TOL group are involved in several signal transduction cascades such as members of the MAPKKK cascade (IL6, Tob2, Stk39, and Dusp24). Other genes involved in lymphocyte differentiation and highly expressed in the TOL group are lymphotactin, the estrogen receptors (ERs) and the suppressor of cytokine signaling 7. Common transcription factors such as ER 1 alpha, GATA-binding protein 1, insulin promoter factor 1, and paired-related homeobox 2 could be identified in the promoter regions of upregulated genes in the TOL group. These data suggest that T lymphoctes of tolerant mice exhibit a distinct molecular expression profile, which needs to be evaluated in other experimental tolerance models to determine whether it is a universal signature of tolerance.

Animals↗
PubMed2005

High-pressure studies of the reaction mechanism of nitric-oxide synthase.

Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N(omega)-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.

Animals↗
PubMed2005

Quantitative analysis of protein dynamics during asymmetric cell division.

In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis.

Animals↗
PubMed2005

Detection of coregulation in differential gene expression profiles.

Genomics and proteomics approaches generate distinct gene expression and protein profiles, listing individual genes embedded in broad functional terms as gene ontologies. However, interpretation of gene profiles in a regulatory and functional context remains a major issue. Elucidation of regulatory mechanisms at the gene expression level via analysis of promoter regions is a prominent procedure to decipher such gene regulatory networks. We propose a novel genetic algorithm (GA) to extract joint promoter modules in a set of coexpressed genes as resulting from differential gene expression experiments. Algorithm design has focused on the following constraints: (I) identification of the major promoter modules, which are (II) characterized by a maximum number of joint motifs and (III) are found in a maximum number of coexpressed genes. The capability of the GA in detecting multiple modules was evaluated on various test data sets, analyzing the impact of the number of motifs per promoter module, the number of genes associated with a module, as well as the total number of distinct promoter modules encoded in a sequence set. In addition to the test data sets, the GA was evaluated on two biological examples, namely a muscle-specific data set and the upstream sequences of the beta-actin gene (ACTB) derived from different species, complemented by a comparison to alternative promoter module identification routines.

Actins↗
PubMed2005

Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system.

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.

Animals↗
PubMed2005

Effects of nitroglycerin/L-cysteine on soluble guanylate cyclase: evidence for an activation/inactivation equilibrium controlled by nitric oxide binding and haem oxidation.

GTN (nitroglycerin; glycerol trinitrate) causes dilation of blood vessels via activation of nitric oxide (NO)-sensitive sGC (soluble guanylate cyclase), a heterodimeric haem protein that catalyses the conversion of GTP into cGMP. Activation of sGC by GTN requires enzymatic or non-enzymatic bioactivation of the nitrate. Based on insufficient NO release and lack of spectroscopic evidence for formation of NO-sGC, the cysteine (Cys)-dependent activation of sGC by GTN was proposed to occur in an NO-independent manner. This extraordinary claim is questioned by the present findings. First, the effect of GTN/Cys was blocked by the NO scavenger oxyhaemoglobin, the superoxide-generating compound flavin mononucleotide and the haem-site sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Secondly, at equi-effective concentrations, GTN/Cys and the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine released identical amounts of NO. Finally, at sufficiently high rates of NO release, activation of sGC by GTN/Cys was accompanied by a shift of the Soret band from 431 to 399 nm, indicating formation of NO-sGC. In the absence of Cys, GTN caused haem oxidation, apparent as a shift of the Soret band to 392 nm, which was accompanied by inactivation of the NO-stimulated enzyme. These results suggest that the effect of GTN/Cys is the result of an activation/inactivation equilibrium that is controlled by the rate of NO release and haem oxidation.

Animals↗
PubMed2005

Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide.

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.

Aldehyde Dehydrogenase↗
PubMed2005

Tetrahydrobiopterin as combined electron/proton donor in nitric oxide biosynthesis: cryogenic UV-Vis and EPR detection of reaction intermediates.

The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the object of intense research in the last few years. It was found that in addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II).O2] heme that is formed as an intermediate in the catalytic cycle. Cryogenic spectroscopic techniques proved particularly useful in the identification of this role of BH4 in NO synthesis. With these methods, the mechanism of fast reactions, such as the reaction of ferrous NOS with O2, can be unraveled by lowering the reaction temperature to subzero values. This may not only reduce the rate to such an extent that the reaction can be followed on a time scale from seconds to minutes, but intermediates may be observed that do not accumulate at higher temperatures. Cryogenic ultraviolet-visible (UV-vis) and electron paramagnetic resonance spectroscopy have been applied to clarify why the BH4 analogue 4-amino-tetrahydrobiopterin (4-amino-BH4) is unable to support NO synthesis. In the course of these studies, evidence was gathered supporting a role for BH4 as an obligate proton and electron donor. It is believed that the inhibitory action of 4-amino-BH4 derives from an inability to serve as a proton donor, even though it is perfectly able to serve as an electron donor. In this chapter, the suitability, drawbacks, and advantages of cryogenic methods are discussed.

Biopterins↗
PubMed2005

Transcriptional response in the unaffected kidney after contralateral hydronephrosis or nephrectomy.

BACKGROUND: Unilateral loss of kidney function is followed by compensatory contralateral growth. The early, genome-wide transcriptional response of the untouched kidney to unilateral ureteral obstruction (UUO) or unilateral nephrectomy is unknown. METHODS: Twelve adult male Sprague-Dawley rats were subjected to UUO and twelve rats to unilateral nephrectomy. At time points 12, 24, and 72 hours after insult four rats each were sacrificed and the contralateral kidney harvested for genome-wide gene expression analysis, transcription factor analysis, and histomorphology. RESULTS: Microarray studies revealed that the majority of differentially expressed transcripts were suppressed in UUO and unilateral nephrectomy compared to control kidneys. The function of these suppressed genes is predominantly growth inhibition and apoptosis suggesting a net pro-hypertrophic response. Insulin-like growth factor-2 (IGF-2)-binding protein was one of the few activated genes. We observed a distinctly different molecular signature between UUO and unilateral nephrectomy at the three time points investigated. The early response in UUO rats suggests a counterbalance to the nonfiltering kidney by activation of transport pathways such as the aquaporins. Unilateral nephrectomy kidneys, on the other hand, respond immediately to contralateral nephrectomy by activation of cell cycle regulators such as the cyclin family. Several genes with weakly defined function were found to be associated with either UUO or unilateral nephrectomy. Transcription factor analysis of the identified transcripts suggests common regulation at least of some of these genes. All kidneys showed normal histology. CONCLUSION: Release of growth inhibition by nephrectomy leads to immediate cell cycle activation after unilateral nephrectomy, whereas UUO kidneys counterbalance filtration failure by activation of several transporters.

Animals↗
PubMed2004

Tetrahydrobiopterin binding to aromatic amino acid hydroxylases. Ligand recognition and specificity.

The three aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylase) and nitric oxide synthase (NOS) all utilize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) as cofactor. The pterin binding site in the three hydroxylases is well conserved and different from the binding site in NOS. The structures of phenylalanine hydroxylase (PAH) and of NOS in complex with BH(4) are still the only crystal structures available for the reduced cofactor-enzyme complexes. We have studied the enzyme-bound and free conformations of BH(4) by NMR spectroscopy and molecular docking into the active site of the three hydroxylases, using endothelial NOS as a comparative probe. We have found that the dihydroxypropyl side chain of BH(4) adopts different conformations depending on which hydroxylase it interacts with. All the bound conformations are different from that of BH(4) free in solution at neutral pH. The different bound conformations appear to result from specific interactions with nonconserved amino acids at the BH(4) binding sites of the hydroxylases, notably the stretch 248-251 (numeration in PAH) and the residue corresponding to Ala322 in PAH, i.e., Ser in TH and Ala in TPH. On the basis of analysis of molecular interaction fields, we discuss the selectivity determinants for each hydroxylase and explain the high-affinity inhibitory effect of 7-tetrahydrobiopterin specifically for PAH.

Amino Acid Sequence↗
PubMed2004

Consumption of nitric oxide by endothelial cells: evidence for the involvement of a NAD(P)H-, flavin- and heme-dependent dioxygenase reaction.

In the present study, we investigated the mechanism of nitric oxide (NO) inactivation by endothelial cells. All experiments were performed in the presence of superoxide dismutase to minimize the peroxynitrite reaction. Incubation of the NO donor diethylamine/NO adduct with increasing amounts of intact cells led to a progressive decrease of the NO concentration, demonstrating a cell-dependent consumption of NO. In cell homogenates, consumption of NO critically depended on the presence of NADPH or NADH and resulted in the formation of nitrate. Both NO consumption and nitrate formation were largely inhibited by the heme poisons NaCN and phenylhydrazine as well as the flavoenzyme inhibitor diphenylene iodonium. Further characterization of this NO consumption pathway suggests that endothelial cells express a unique membrane-associated enzyme or enzyme system analogous to the bacterial NO dioxygenase that converts NO to nitrate in a NAD(P)H-, flavin- and heme-dependent manner.

Animals↗
PubMed2004

Interference of the polyphenol epicatechin with the biological chemistry of nitric oxide- and peroxynitrite-mediated reactions.

The formation of reactive nitrogen species in mammalians has both beneficial and undesirable effects. Nitric oxide (NO) production in endothelial cells leads to vascular smooth muscle relaxation, but if reactive nitrogen species are generated in high amounts by cells under inflammatory conditions they are toxic. Flavonoids like (-)-epicatechin show an inverse association of their intake with diseases thought to be associated with overproduction of reactive nitrogen species. We found that the formation of cyclic GMP in cultured porcine aortic endothelial cells was not affected by up to 1 mM (-)-epicatechin. Half maximal inhibition of interferon-gamma/lipopolysaccharide induced nitrite accumulation in murine macrophages required about 0.5 mM of the flavonoid. In contrast, nitration of free tyrosine triggered by 0.1 and 1 mM authentic peroxynitrite was inhibited by (-)-epicatechin with IC(50) values of 6.6 and 28.0 microM, respectively. The presence of 15 mM sodium bicarbonate had no significant effect. Nitration of protein-bound tyrosine in phorbol 12-myristate 13-acetate treated HL-60 cells in the presence of nitrite was inhibited by (-)-epicatechin at a similar concentration range (IC(50)=10-100 microM). Myeloperoxidase activity of phorbol 12-myristate 13-acetate stimulated HL-60 cells was inhibited by (-)-epicatechin with an IC(50) value of 77.4 microM. Epicatechin inhibited dihydrorhodamine oxidation by 50 microM authentic peroxynitrite and 1 mM 3-morpholino-sydnonimine with IC(50) values of 11.8 and 0.63 microM, respectively. Our data suggest that at up to 0.1 mM (-)-epicatechin preferentially inhibits NO-related nitration and oxidation reactions without affecting NO synthesis and cyclic GMP signaling.

Catechin↗