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Amphetamines and cocaine are illicitly used psychostimulants which act on monoaminergic neurotransmitter transporters. These comprise the transporters for dopamine, serotonin, or norepinephrine. By themselves, the drugs can already cause... more
Amphetamines and cocaine are illicitly used psychostimulants which act on monoaminergic neurotransmitter transporters. These comprise the transporters for dopamine, serotonin, or norepinephrine. By themselves, the drugs can already cause severe problems in terms of neurotoxicity and addiction. Nevertheless, additional health threats arise from the possibility that these compounds are cut by adding adulterant substances without declaration. This chapter examines commonly found adulterants of drugs which are sold as cocaine on the street market. The typical pharmacological profile is discussed and the chapter elaborates on reasons why these compounds can be used as cutting agents. It has been found that a subset of these adulterants exerts effects similar to cocaine itself. Furthermore, we will discuss the example of levamisole, which is the most frequently used cocaine cutting agent today, and its metabolite aminorex.
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Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs and xenobiotics. We previously reported novel missense mutations of organic cation transporter 3 (OCT3, SLC22A3), some with drastically... more
Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs and xenobiotics. We previously reported novel missense mutations of organic cation transporter 3 (OCT3, SLC22A3), some with drastically impacted transport capabilities compared to wildtype. For some variants, this was due to ER retention and subsequent degradation of the misfolded transporter. For other transporter families, it was previously shown that treatment of misfolded variants with pharmacological and chemical chaperones could restore transport function to a certain degree. To investigate two potentially ER-bound, misfolded variants (D340G and R348W), we employed confocal and biochemical analyses. In addition, radiotracer uptake assays were conducted to assess whether pre-treatment with chaperones could restore transporter function. We show that pre-treatment of cells with the chemical chaperone 4-PBA (4-phenyl butyric acid) leads to increased membrane expression of misfolded var...
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The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle.... more
The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives) and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted ...
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Objectives: Reimbursement decisions on new medicines require an assessment of their value. In Austria, when applying for reimbursement of new medicines, pharmaceutical companies are also obliged to submit forecasts of future sales. We... more
Objectives: Reimbursement decisions on new medicines require an assessment of their value. In Austria, when applying for reimbursement of new medicines, pharmaceutical companies are also obliged to submit forecasts of future sales. We systematically examined the accuracy of these pharmaceutical sales forecasts and hence the usefulness of these forecasts for reimbursement evaluations. Methods: We retrospectively analyzed reimbursement applications of 102 new drugs submitted between 2005 and 2014, which were accepted for reimbursement outside of hospitals, and for which actual reimbursed sales were available for at least 3 years. The main outcome variable was the accuracy ratio, defined as the ratio of forecasted sales submitted by pharmaceutical companies when applying for reimbursement to actual sales from reimbursement data. Results: The median accuracy ratio [95% confidence interval] was 1.33 [1.03; 1.74, range 0.15–37.5], corresponding to a median overestimation of actual sales b...
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The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, for... more
The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K+ gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP+(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of...
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Lead exposure can cause substantial organ damage. Enteral lead absorption may be reduced by concomitant intake of clinoptilolite tuff, a zeolite from natural sources. This study aimed to assess the effect of purified clinoptilolite tuff... more
Lead exposure can cause substantial organ damage. Enteral lead absorption may be reduced by concomitant intake of clinoptilolite tuff, a zeolite from natural sources. This study aimed to assess the effect of purified clinoptilolite tuff (G-PUR) on enteral lead uptake in adults using stable lead isotope 204Pb as a tracer. In this randomized, placebo-controlled, double-blind, parallel-group study, 42 healthy participants were randomized to receive oral G-PUR 2.0 g, 2 * 2.0 g, or placebo, together with 2.5 µg of 204Pb in water. The enrichment of 204Pb caused by the tracer in blood and urine was measured by mass spectrometry. G-PUR was well tolerated. The mean maximum 204Pb enrichment of 0.505% of total blood lead was significantly higher (p < 0.0001) in the placebo group compared to G-PUR 2.0 g (0.073%) or G-PUR 2 * 2.0 g (0.057%) group. Normalized 204Pb AUC0-192 was 86.5, 11.9, and 8.5% * h without and with G-PUR 2.0 g, and G-PUR 2 * 2.0 g, respectively (p < 0.0001 vs. placebo)....
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Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (GPCR) drug discovery and development. Peptides occupy a unique chemical space that is not easily accessible for small molecules and... more
Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (GPCR) drug discovery and development. Peptides occupy a unique chemical space that is not easily accessible for small molecules and antibodies and provide advantages over these ligand classes such as lower toxicity and higher selectivity. The κ-opioid receptor (KOR) is a prototypic GPCR and an appealing therapeutic target for the development of safer and more effective analgesics. Recently, peptides have emerged as analgesic drug candidates with improved side effect profiles. We have previously identified plant-derived peptides, which activate KOR. Based on this precedent, here we relied on publicly available databases to discover novel KOR peptide ligands by genome mining. Using human preprodynorphin as a query, we identified blenny fish-derived peptides, referred to as blenniorphins, capable of binding to and activating KOR with nanomolar affinity and potency, respectively. Additionall...
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Viral vectors restore dopamine transporter function and ameliorate neuropathology in iPSC-derived neurons and a mouse model of infantile parkinsonism.
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We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and... more
We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.
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Research Interests: Chemistry, Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreAdenosine, Brain, Humans, GPCRs, Escherichia coli, Animals, Biological, CHEMICAL SCIENCES, Cattle, Adenosine Receptors, Base Sequence, G Proteins, Gtp Binding Proteins, Adenosine receptor, and Medical and Health Sciences
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The dopamine transporter (DAT) retrieves dopamine into presynaptic terminals after synaptic release. The concentrative power of DAT is thought to be fueled by the transmembrane Na+gradient, but it is conceivable that DAT can also rely on... more
The dopamine transporter (DAT) retrieves dopamine into presynaptic terminals after synaptic release. The concentrative power of DAT is thought to be fueled by the transmembrane Na+gradient, but it is conceivable that DAT can also rely on other energy sources, e.g. membrane voltage and/or the K+gradient. Here, we recorded uptake of dopamine or the fluorescent substrate APP+((4-(4-dimethylamino)phenyl-1-methylpyridinium) in DAT-expressing cells under voltage control. We show that DAT differs substantially from the closely related serotonin transporter (SERT): substrate uptake by DAT was voltage-dependent, intracellular K+binding to DAT was electrogenic but transient in nature thus precluding antiport of K+by DAT. There is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power. Based on our observations, we conclude that subtle differences in the kinetics of co-substrate ion binding allow closely related transporters to select between ...
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Flunixin is a nonsteroidal anti‐inflammatory drug (NSAID) that has anti‐inflammatory, anti‐pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne® Transdermal, MSD Animal Health) and is now the... more
Flunixin is a nonsteroidal anti‐inflammatory drug (NSAID) that has anti‐inflammatory, anti‐pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne® Transdermal, MSD Animal Health) and is now the first NSAID registered to be administered as a pour‐on product in cattle. According to the manufacturer's instructions, the pour‐on product should be applied only to dry skin and exposure to rain should be avoided for at least 6 hr after application. The objective of the study was to evaluate the effect of simulated exposure to light or heavy rain on flunixin absorption and bioavailability within the first 4 hr after administration. Therefore, an isocratic HPLC method was developed to quantify flunixin concentrations in bovine serum by UV detection. Light rain decreased flunixin absorption only when rain started immediately after flunixin administration, while light rain starting more than 30 min after administration of flunixin had no effect on ...
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Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl−. Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and... more
Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl−. Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl− in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl− on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstra...
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Research Interests: Psychology, Chemistry, Kinetics, Neuropharmacology, Medicine, and 15 moreMolecular modeling, Serotonin Transporter, Amphetamine, Neurotransmitters, Mephedrone, Methcathinone, Dopamine Transporter, Allosteric modulators, Chemical Synthesis, Cathinones, Neurosciences, Neurotransmitter Transporters, Patch Clamp, Allosteric regulation, and Pharmacology and pharmaceutical sciences
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Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible... more
Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum (ER) when heterologously expressed in HEK293 cells. In three of these mutants - i.e. hDAT-V158F, hDAT-G327R and hDAT-L368Q- the folding deficit was remedied with the pharmacochaperonenoribogaineor the heat shock protein 70 (HSP70)-inhibitor pifithrin-μ such that ER export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore, whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or ...
Research Interests: Animal Behavior, Biology, Drosophila melanogaster, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Dopamine, Humans, Mutation, Animals, Male, Endoplasmic Reticulum, Ibogaine, CHEMICAL SCIENCES, Dopamine Transporter, Juvenile, Dopaminergic Neurons, dopaminergic, parkinsonian disorders, and Medical and Health Sciences
Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was... more
Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially ...
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Research Interests: Molecular Dynamics Simulation, Protein Folding, Biology, Drosophila melanogaster, Cell Biology, and 15 moreBiological Chemistry, Medicine, Biological Sciences, Dopamine, Humans, Mutation, Animals, Endoplasmic Reticulum, Sleep, Ibogaine, Phenotype, CHEMICAL SCIENCES, Dopamine Transporter, dopaminergic, and Medical and Health Sciences
Research Interests: Cell Cycle, Survival Analysis, Molecular Pharmacology, Medicine, Transplantation, and 15 moreHematopoietic Stem Cells, Cell Differentiation, Humans, cyclic AMP, Animals, Bone marrow, Haematopoiesis, Bone Marrow Transplantation, DRUG REPOSITIONING, Cell Survival, Neurosciences, Hematopoietic Stem Cell Transplantation, epoprostenol, Pharmacology and pharmaceutical sciences, and Cholera toxin
We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with... more
We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a recept...
Research Interests: Neuroscience, Biology, Drug interactions, Medicine, Fluorescence Resonance Energy Transfer, and 15 moreBrain, Humans, Mice, Animals, Potassium, Neurons, Astrocytes, Long Term Depression, Immunoprecipitation, Analysis of Variance, Glial Fibrillary Acidic Protein, Hydrolysis, Glutamate Receptor, coculture techniques, and Medical and Health Sciences
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The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in... more
The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388–400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.
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ZusammenfassungApotheker und hausapothekenführende Ärzte aufgepasst: Die Nichteinhaltung der Kühlkette bei der Lagerung von biologisch hergestellten Arzneimitteln (Biologicals und Biosimilars) birgt ein hohes Haftungspotenzial! Neben der... more
ZusammenfassungApotheker und hausapothekenführende Ärzte aufgepasst: Die Nichteinhaltung der Kühlkette bei der Lagerung von biologisch hergestellten Arzneimitteln (Biologicals und Biosimilars) birgt ein hohes Haftungspotenzial! Neben der vertraglichen Pflichtverletzung kann es zur Verletzung eines Schutzgesetzes, zu verwaltungsrechtlichen Sanktionen bis hin zur strafrechtlichen Verurteilung kommen. Abhilfe kann eine lückenlose und langfristige Dokumentation der Kühlkette bringen.AbstractPharmacists and physicians running dispensaries beware: storage of biopharmaceuticals (biologics and biosimilars) is contingent on an adequate cold chain and thus bears a considerable liability risk! The contractual neglect of duty aside, this comprises the breach of a protection law and the risk of being held liable under administrative law and/or of a criminal conviction. A prudent remedial measure is to provide a complete documentation of the cold chain and to assure long-term accessibility of the pertinent data.
Research Interests: Medicine and Biosimilar
Research Interests: Chemistry, Protein Folding, Biological Chemistry, Medicine, Biological Sciences, and 12 moreMolecular chaperones, Serotonin, Humans, Serotonin Transporter, CHEMICAL SCIENCES, Sodium, Transporter, Protein Conformation, Lipid bilayers, Ion Transport, Amino Acid Substitution Rates, and Medical and Health Sciences
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biological properties in immunology and oncology. Circulating MIF levels are elevated... more
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biological properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both, stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01 and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase (ERK1/2) and protein ki...
Chaperoning of the A1-adenosine receptor by endogenous adenosine – an extension of the retaliatory metabolite concept
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Plasmalemmal solute carriers (SLCs) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino... more
Plasmalemmal solute carriers (SLCs) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino acid sequence that often leads to impaired protein function and onset of monogenic disorders. To understand how these altered proteins cause disease, it is necessary to undertake relevant functional assays. These experiments reveal descriptors of SLC function such as the maximal transport velocity (Vmax), the Michaelis constant for solute uptake (KM), potencies for inhibition of transporter function (IC50/EC50), and many more. In several instances, the mutated versions of different SLC transporters differ from their wild-type counterparts in the value of these descriptors. While determination of these experimental parameters can provide conjecture as to how the mutation gives rise to disease, they seldom provide any definitive insights on how a vari...
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Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate... more
Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding p...
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Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not... more
Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not delivered to the cell surface because it is retained in the endoplasmic reticulum (ER). The underlying defect is improper folding of the transporter and is the case for many of the known dopamine transporter mutants. The monoamine transporters, i.e. the transporters for norepinephrine (NET/SLC6A2), dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4), have a rich pharmacology; hence, their folding-deficient mutants lend themselves to explore the concept of pharmacological chaperoning. Pharmacochaperones are small molecules, which bind to folding intermediates with exquisite specificity and scaffold them to a folded state, which is exported from the ER and delivered to the cell surface. Pharmacochaperoning of mutant monoamine transporters, however, is not...
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The brain of Drosophila melanogaster is comprised of some 100,00 neurons, 127 and 80 of which are dopaminergic and serotonergic, respectively. Their activity regulates behavioral functions equivalent to those in mammals, e.g. motor... more
The brain of Drosophila melanogaster is comprised of some 100,00 neurons, 127 and 80 of which are dopaminergic and serotonergic, respectively. Their activity regulates behavioral functions equivalent to those in mammals, e.g. motor activity, reward and aversion, memory formation, feeding, sexual appetite etc. Mammalian dopaminergic and serotonergic neurons are known to be heterogeneous. They differ in their projections and in their gene expression profile. A sophisticated genetic tool box is available, which allows for targeting virtually any gene with amazing precision in Drosophila melanogaster. Similarly, Drosophila genes can be replaced by their human orthologs including disease-associated alleles. Finally, genetic manipulation can be restricted to single fly neurons. This has allowed for addressing the role of individual neurons in circuits, which determine attraction and aversion, sleep and arousal, odor preference etc. Flies harboring mutated human orthologs provide models, w...
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Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin.... more
Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [125I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC50 = 4.9±1.2µM and 19.9±1.8µM, respectively) and blocked the cross-linking of [125I]calmodulin to some, but not all, target proteins in brain membranes by [125I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein βγ dimers (Gβγ) from porcine brain and a glutathione S-transferase-fusion protein co...
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Cocaine and amphetamine are psychostimulant drugs that are illicitly used; they affect sensory perception by targeting the neurotransmitter: sodium symporters (NSS) at the synapses between neurons. They both increase the concentration of... more
Cocaine and amphetamine are psychostimulant drugs that are illicitly used; they affect sensory perception by targeting the neurotransmitter: sodium symporters (NSS) at the synapses between neurons. They both increase the concentration of the neurotransmitter in the synaptic cleft but by different means.
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The McCune-Albright syndrome (MAS) comprises a triad of physical signs: localized bone lesions termed polyostotic fibrous dysplasia, café-au-lait pigmentation of the skin, and autonomous hyperfunction of multiple endocrine systems,... more
The McCune-Albright syndrome (MAS) comprises a triad of physical signs: localized bone lesions termed polyostotic fibrous dysplasia, café-au-lait pigmentation of the skin, and autonomous hyperfunction of multiple endocrine systems, including overproduction of GH and T4. A somatic activating point mutation in the gene for the alpha-subunit of the G-protein (Gs alpha) in the affected tissue has been claimed to be the underlying defect. A 29-yr-old patient with MAS, showing polyostotic fibrous dysplasia associated with acromegalic features, underwent endocrinological studies, including oral glucose tolerance test and pituitary stimulation test, and magnetic resonance imaging, revealing elevated plasma concentrations of GH, PRL, and secondary hyperthyroidism due to pituitary macroadenoma infiltrating the sphenoid cavity and extending to the suprasellar space. Subsequently, reduction of tumor mass by a transsphenoidal and a subsequent subfrontal operation led to only marginal amelioration of the excessive hormone production. Postsurgery octreotide and bromocriptine therapy induced near-normalization of hormone concentrations. Immunohistochemistry of tumor tissue confirmed the plurihormonal character, but DNA sequence analysis did not detect any of the two known activating mutations in the Gs alpha gene. Furthermore, biochemical tests revealed normal Gs alpha function, ruling out other mutations that lead to constitutive Gs alpha activation. Our study documents that MAS is a heterogeneous disease. Some, but clearly not all, patients have oncogenic mutations of the gene coding for Gs alpha. Any gene acting down-stream of Gs can theoretically be predicted to result in the same phenotype. In addition, hyperthyroidism of MAS may be secondary to a TSH-producing pituitary macroadenoma.
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Research Interests: Chemistry, Electrophysiology, Molecular Dynamics Simulation, Biology, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Humans, Mutation, Animals, Biological, Phosphorylation, CHEMICAL SCIENCES, Protein Conformation, Amphetamines, Amino Acid Sequence, Oocytes, Molecular Sequence Data, Cell Membrane, and Medical and Health Sciences
The annexation of Austria by Nazi Germany from 1938 to 1945, or “Anschluss”, was the darkest chapter in the country’s history. The contributions presented in this paper demonstrate that we still feel the aftermath of this horrible period.... more
The annexation of Austria by Nazi Germany from 1938 to 1945, or “Anschluss”, was the darkest chapter in the country’s history. The contributions presented in this paper demonstrate that we still feel the aftermath of this horrible period. It was horrible not only because Austria was a victim of Nazi terror, but, moreover, was a perpetrator of it. While invading Austria, poorly prepared German troops were surprised to be received with cheering crowds, much less the overcrowded Heldenplatz during Hitler’s speech to the Austrian population on 15 March 1938. Everything was well prepared for the arrival of the German occupiers: already the years before, subsidiaries of the NSDAP were active in Austria, and there were suddenly hundreds of thousands of swastikas and flags available and an extreme and sophisticated system of denunciation. Many Austrians, including doctors, achieved leading positions during the Nazi period. Doctors represented the highest proportion of Austrian academics who were members of the NSDAP (though it is worth noting that many of their Jewish colleagues had already been expelled from the country), and they were heavily implicated in committing ethical misconduct, in particular in the execution of the “euthanasia” T4-programme, where handicapped children and adults were killed. After World War II, many tainted physicians and university professors were reinstated in their former positions and had the opportunity of a post-war career. This was the main reason for the general backlog in research and development in Austria in comparison with most countries of the Western world.
Volltext: https://link.springer.com/content/pdf/10.1007/s00508-018-1366-4.pdf
Volltext: https://link.springer.com/content/pdf/10.1007/s00508-018-1366-4.pdf