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Biomedical subjects

Peter M Schmidt

Publications and source records attributed to Peter M Schmidt.

11 recordsLinked to original sources
PubMed2006

Identification of residues crucially involved in soluble guanylate cyclase activation.

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.

Animals↗
PubMed2006

The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells.

Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.

Adult↗
PubMed2006

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential.

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

Animals↗
PubMed2006

Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

Animals↗
PubMed2005

Residues stabilizing the heme moiety of the nitric oxide sensor soluble guanylate cyclase.

Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.

Alanine↗
PubMed2004

Oligonucleotide microarray for identification of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA.

We developed a DNA microarray for identification of Bacillus anthracis and other phylogenetic groupings within the "Bacillus cereus group". Nucleotide sequences of 16S-23S ribosomal DNA internal transcribed spacers containing genes for tRNA(Ile) from 52 B. anthracis strains were found to be identical to sequences from seven strains published previously and different from all other bacteria. When 42 oligonucleotide probes targeting polymorphic sites were immobilized on glass slides and hybridized to fluorescently labeled PCR amplification products, one or more mismatches could be discriminated in all but one cases. Hence, hybridization events were highly specific and identification of B. anthracis was straightforward.

Bacillus anthracis↗
PubMed2003

Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase.

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.

Amino Acid Sequence↗
PubMed2002

Detection of activity of telomerase in tumor cells using fiber optical biosensors.

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.

Biomarkers, Tumor↗
PubMed2002

Real-time determination of telomerase activity in cell extracts using an optical biosensor.

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.

Base Sequence↗
PubMed2001

Determination of the turnover number of the restriction endonuclease EcoRI using evanescent wave technology.

Binding and catalytic activity of the type II restriction endonuclease EcoRI on immobilized DNA has been observed in real time using three different evanescent wave biosensors and two different immobilization techniques. The method gives direct access to the turnover number (kcat) without the necessity for the determination of any concentration or activity. The combination of different evanescent wave techniques gives access to the catalytic mechanism and allows the determination of the rate limiting step.

Biosensing Techniques↗