Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis.

Aim: The aim of our study was to explore the pathophysiologic role of oxidation of hemoglobin (Hb) to ferrylHb in human atherosclerosis. Results: We observed a severe oxidation of Hb to ferrylHb in complicated atherosclerotic lesions of carotid arteries with oxidative changes of the globin moieties, detected previously described oxidation hotspots in Hb (ß1Cys93; ß1Cys112; ß2Cys112) and identified a novel oxidation hotspot (α1Cys104). After producing a monoclonal anti-ferrylHb antibody, ferrylHb was revealed to be localized extracellularly and also internalized by macrophages in the human hemorrhagic complicated lesions. We demonstrated that ferrylHb is taken up via phagocytosis as well as CD163 receptor-mediated endocytosis and then transported to lysosomes involving actin polymerization. Internalization of ferrylHb was accompanied by upregulation of heme oxygenase-1 and H-ferritin and accumulation of iron within lysosomes as a result of heme/iron uptake. Importantly, macrophages exposed to ferrylHb in atherosclerotic plaques exhibited a proinflammatory phenotype, as reflected by elevated levels of IL-1ß and TNF-α. To find further signatures of ferrylHb in complicated lesions, we performed RNA-seq analysis on biopsies from patients who underwent endarterectomies. RNA-seq analysis demonstrated that human complicated lesions had a unique transcriptomic profile different from arteries and atheromatous plaques. Pathways affected in complicated lesions included gene changes associated with phosphoinositide 3-kinase (PI3K) signaling, lipid transport, tissue remodeling, and vascularization. Targeted analysis of gene expression associated with calcification, apoptosis, and hemolytic-specific clusters indicated an increase in the severity of complicated lesions compared with atheroma. A 39% overlap in the differential gene expression profiles of human macrophages exposed to ferrylHb and the complicated lesion profiles was uncovered. Among these 547 genes, we found inflammatory, angiogenesis, and iron metabolism gene clusters regulated in macrophages. Innovation and Conclusion: We conclude that oxidation of Hb to ferrylHb contributes to the progression of atherosclerosis via polarizing macrophages into a proatherogenic phenotype. Antioxid. Redox Signal. 35, 917-950.

On the early events of the calcium-induced activation of coagulation factor XIII-A2 and tissue transglutaminase: an in silico study.

Both coagulation factor XIII-A2 (FXIII-A2) and tissue transglutaminase (TG2) play distinctive and important roles in homeostasis by crosslinking proteins or peptides via isopeptide bonds. In this present study, a series of microsecond-long all-atom molecular dynamics (MD) simulations were carried out in order to reveal the dynamic, atomic-level events which may contribute to the activation of these proteins via the binding of calcium ions. In addition to previously conducted in vitro and crystallographic studies, further suggestions have been made concerning the calcium binding features of these enzymes. The different systems used for running the simulations were based on the zymogen, computationally cleaved and even the activation peptide (AP-FXIII) free FXIII-A2' homodimer form. The effects of various ionic environments have also been explored in the simulations of FXIII-A2. Our results suggest that the presence of calcium ions can cause increased AP fluctuations, which ultimately could lead to their relocation on the homodimer surface. The release of these APs seems to be crucial for rotation of the A subunits based on equilibrium MD simulations. The primary evidence for this assumption comes from the predicted principal component eigenvector which is considered as the first, large-scale event of the overall activation process. To get a more accurate atomic-level description, the calcium binding sites of TG2 have also been investigated in our extensive in silico experiments, which suggests the presence of previously unidentified binding sites as well.Communicated by Ramaswamy H. Sarma.

The mechanism of high affinity pentasaccharide binding to antithrombin, insights from Gaussian accelerated molecular dynamics simulations.

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from 'static' structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a 'non-activated' AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. 'Generalized correlation' matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.Communicated by Ramaswamy H. Sarma.

First Step of the Transglutaminase Reaction Catalyzed by Activated Factor XIII Subunit A, Hybrid Quantum Chemistry/Molecular Mechanics Calculations.

The A subunit of blood coagulation factor XIII belongs to the family of transglutaminase enzymes. Its active form (FXIIIa) catalyzes isopeptide bond formation between Glu and Lys residues of specific substrates. Little data are available on the mechanism of this reaction. In this work, the first step of the proposed two-step process was investigated using two different protocols of hybrid QM/molecular mechanics (MM) calculations: an ONIOM-based model as well as QM/MM/molecular dynamics (MD) metadynamics simulations in explicit TIP3P solvent with Gromacs, PLUMED, and a DFTB3 package. Based on calculations involving a truncated system derived from docking of a peptide substrate, our study confirms the higher stability of a zwitterionic form of the catalytic Cys and His residues in the Michaelis complex as well as the "resting" state of the enzyme. Potential energy surfaces, obtained by geometry optimizations with Gaussian, show a two-step reaction mechanism with a zwitterionic tetrahedral intermediate formation in the first and NH3 dissociation in the second step in the case of our ONIOM system. In contrast, in QM/MM MD metadynamics simulations, all three steps occurred in a concerted manner. As a conclusion, our model is able to provide insights into the reaction mechanism of this enzyme.

Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations.

Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.

Perspectives of a myosin motor activator agent with increased selectivity.

Clinical treatment of heart failure is still not fully solved. A novel class of agents, the myosin motor activators, acts directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. Omecamtiv mecarbil, the lead molecule of this group, is now in human phase 3 displaying promising clinical performance. However, omecamtiv mecarbil is not selective to myosin, because it readily binds to and activates cardiac ryanodine receptors (RyR-2), an effect that may cause complications in case of overdose. In this study, in silico analysis was performed to investigate the docking of omecamtiv mecarbil and other structural analogues to cardiac myosin heavy chain and RyR-2 to select the structure that has a higher selectivity to myosin over RyR-2. In silico docking studies revealed that omecamtiv mecarbil has comparable affinity to myosin and RyR-2: the respective Kd values are 0.60 and 0.87 µmol/L. Another compound, CK-1032100, has much lower affinity to RyR-2 than omecamtiv mecarbil, while it still has a moderate affinity to myosin. It was concluded that further research starting from the chemical structure of CK-1032100 may result a better myosin activator burdened probably less by the RyR-2 binding side effect. It also is possible, however, that the selectivity of omecamtiv mecarbil to myosin over RyR-2 cannot be substantially improved, because similar moieties seem to be responsible for the high affinity to both myosin and RyR-2.

Omecamtiv Mecarbil: A Myosin Motor Activator Agent with Promising Clinical Performance and New in vitro Results.

BACKGROUND: Clinical treatment of heart failure is still suffering from limited efficacy and unfavorable side effects. The recently developed group of agents, the myosin motor activators, act directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. The lead molecule, omecamtiv mecarbil is now in human 3 stage. In addition to the promising clinical data published so far, there are new in vitro results indicating that the effect of omecamtiv mecarbil on contractility is rate-dependent. Furthermore, omecamtiv mecarbil was shown to activate cardiac ryanodine receptors, an effect that may carry proarrhythmic risk. METHODS: These new results, together with the controversial effects of the drug on cardiac oxygen consumption, are critically discussed in this review in light of the current literature on omecamtiv mecarbil. RESULTS: In therapeutically relevant concentrations the beneficial inotropic effect of the agent is not likely affected by these new results - in accordance with the good clinical data. At supratherapeutic concentrations, however, activation of cardiac ryanodine receptors may increase arrhythmia propensity, and the stronger effect on diastolic than systolic cell shortening, observed at higher pacing frequencies, may decrease or offset the inotropic effect of omecamtiv mecarbil. CONCLUSION: Further studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects.

Clinical and laboratory characteristics of antithrombin deficiencies: A large cohort study from a single diagnostic center.

INTRODUCTION: Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. PATIENTS AND METHODS: Non-related AT deficient patients (n=156) and their family members (total n=246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. RESULTS: Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. CONCLUSION: Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.

Antithrombin Debrecen (p.Leu205Pro) - Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency.

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS: Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS: Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans­Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS: AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor.

Ca2+ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+'s and Sm3+'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 µM Ca2+, Ln3+ potently inhibited RyR's open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+'s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 µM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further.

Modeling the archetype cysteine protease reaction using dispersion corrected density functional methods in ONIOM-type hybrid QM/MM calculations; the proteolytic reaction of papain.

A proteolytic reaction of papain with a simple peptide model substrate N-methylacetamide has been studied. Our aim was twofold: (i) we proposed a plausible reaction mechanism with the aid of potential energy surface scans and second geometrical derivatives calculated at the stationary points, and (ii) we investigated the applicability of the dispersion corrected density functional methods in comparison with the popular hybrid generalized gradient approximations (GGA) method (B3LYP) without such a correction in the QM/MM calculations for this particular problem. In the resting state of papain the ion pair and neutral forms of the Cys-His catalytic dyad have approximately the same energy and they are separated by only a small barrier. Zero point vibrational energy correction shifted this equilibrium slightly to the neutral form. On the other hand, the electrostatic solvation free energy corrections, calculated using the Poisson-Boltzmann method for the structures sampled from molecular dynamics simulation trajectories, resulted in a more stable ion-pair form. All methods we applied predicted at least a two elementary step acylation process via a zwitterionic tetrahedral intermediate. Using dispersion corrected DFT methods the thioester S-C bond formation and the proton transfer from histidine occur in the same elementary step, although not synchronously. The proton transfer lags behind (or at least does not precede) the S-C bond formation. The predicted transition state corresponds mainly to the S-C bond formation while the proton is still on the histidine Nδ atom. In contrast, the B3LYP method using larger basis sets predicts a transition state in which the S-C bond is almost fully formed and the transition state can be mainly featured by the Nδ(histidine) to N(amid) proton transfer. Considerably lower activation energy was predicted (especially by the B3LYP method) for the next amide bond breaking elementary step of acyl-enzyme formation. Deacylation appeared to be a single elementary step process in all the methods we applied.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 µM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

A few atoms make the difference: synthetic, CD, NMR and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycon derivatives.

Despite the close structural similarity between the heptapeptide cores of the glycopeptide antibiotics teicoplanin and ristocetin, synthetically modified derivatives of their aglycons show significantly different antibacterial and antiviral properties. The teicoplanin aglycon derivatives with one exception proved to be potent antibacterials but they did not exhibit anti-influenza virus activity. In contrast, the aglycoristocetin derivatives generally showed high anti-influenza virus activity and possessed moderate antibacterial activity. A systematic structure-activity relationship study has been carried out on ristocetin and teicoplanin aglycon derivatives, to explore which structural differences are responsible for these markedly different biological activities. According to electronic circular dichroism and in silico conformational studies, it was found that the differences in anti-influenza virus activity are mainly determined by the conformation of the heptapeptide core of the antibiotics controlled by the presence or absence of chloro substituents. Knowledge of the bioactive conformation will help to design new analogs with improved anti-influenza virus activity. For the teicoplanin derivatives, it was shown that derivatization to improve the antiviral efficacy was accompanied by a significant decrease in antibacterial activity.

Molecular characterization of p.Asp77Gly and the novel p.Ala163Val and p.Ala163Glu mutations causing protein C deficiency.

INTRODUCTION: Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates. PATIENTS AND METHODS: The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out. RESULTS AND CONCLUSIONS: The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.

Dynamic properties of the native free antithrombin from molecular dynamics simulations: computational evidence for solvent- exposed Arg393 side chain.

While antithrombin (AT) has small basal inhibitory activity, it reaches its full inhibitory potential against activated blood coagulation factors, FXa, FIXa, and FIIa (thrombin), via an allosteric and/or template (bridging) mechanism by the action of heparin, heparan sulfate, or heparin-mimetic pentasaccharides (PS). From the numerous X-ray structures available for different conformational states of AT, only indirect and incomplete conclusions can be drawn on the inherently dynamic properties of AT. As a typical example, the basal inhibitory activity of AT cannot be interpreted on the basis of "non-activated" free antithrombin X-ray structures since the Arg393 side chain, playing crucial role in antithrombin-proteinase interaction, is not exposed. In order to reveal the intrinsic dynamic properties and the reason of basal inhibitory activity of antithrombin, 2 µs molecular dynamics simulations were carried out on its native free-forms. It was shown from the simulation trajectories that the reactive center loop which is functioning as "bait" for proteases, even without any biasing potential can populate conformational state in which the Arg393 side chain is solvent exposed. It is revealed from the trajectory analysis that the peptide sequences correspond to the helix D extension, and new helix P formation can be featured with especially large root-mean-square fluctuations. Mutual information analyses of the trajectory showed remarkable (generalized) correlation between those regions of antithrombin which changed their conformations as the consequence of AT-PS complex formation. This suggests that allosteric information propagation pathways are present even in the non-activated native form of AT.

Tetrodotoxin blockade on canine cardiac L-type Ca²âº channels depends on pH and redox potential.

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na⁺ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²âº current (I(Ca)) in canine cardiac cells. In the present study, the TTX-sensitivity of I(Ca) was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of I(Ca) in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 µM H2O2). Phosphorylation of the channel protein (induced by 3 µM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 µM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels.

Selectivity problems with drugs acting on cardiac Na⁺ and Ca²âº channels.

With the increase of our knowledge on cardioactive agents it comes more and more clear that practically none of the currently used compounds shows absolute selectivity to one or another ion channel type. This is particularly true for Na(+) and Ca(2+) channel modulators, which are widely applied in the clinical practice and biomedical research. The best example might be probably the marine guanidine poison tetrodotoxin, which has long been considered as a selective Na(+) channel blocker, while recently it turned out to effectively inhibit cardiac Ca(2+) currents as well. In the present study the cross actions observed between the effects of various blockers of Na(+) channels (such as toxin inhibitors, class I antiarrhythmics and local anesthetics) and Ca(2+) channels (like phenylalkylamines, dihydropyridine compounds, diltiazem and mibefradil) are overviewed in light of the known details of the respective channel structures. Similarly, activators of Na(+) channels, including veratridine and batrachotoxin, are also compared. The binding of tetrodotoxin and saxitoxin to Cav1.2 and Nav1.5 channel proteins is presented by construction of theoretical models to reveal common structures in their pore forming regions to explain cross reactions. Since these four domain channels can be traced back to a common ancestor, a close similarity in their structure can well be demonstrated. Thus, the poor selectivity of agents acting on cardiac Na(+) and Ca(2+) channels is a consequence of evolution. As a conclusion, since the limited selectivity is an intrinsic property of drug receptors, it has to be taken into account when designing new cardioactive compounds for either medical therapy or experimental research in the future.

Three novel germ-line VHL mutations in Hungarian von Hippel-Lindau patients, including a nonsense mutation in a fifteen-year-old boy with renal cell carcinoma.

BACKGROUND: Von Hippel-Lindau disease is an autosomal dominantly inherited highly penetrant tumor syndrome predisposing to retinal and central nervous system hemangioblastomas, renal cell carcinoma and phaeochromocytoma among other less frequent complications. METHODS: Molecular genetic testing of the VHL gene was performed in five unrelated families affetced with type I VHL disease, including seven patients and their available family members. RESULTS: Molecular genetic investigations detected three novel (c.163 G > T, c.232A > T and c.555C > A causing p.Glu55X, p.Asn78Tyr and p.Tyr185X protein changes, respectively) and two previously described (c.340 + 1 G > A and c.583C > T, resulting in p.Gly114AspfsX6 and p.195GlnX protein changes, respectively) germline point mutations in the VHL gene. Molecular modeling of the VHL-ElonginC-HIF-1alpha complex predicted that the p.Asn78Tyr amino acid exchange remarkably alters the 77-83 loop structure of VHL protein and destabilizes the VHL-HIF-1alpha complex suggesting that the mutation causes type I phenotype and has high risk to associate to renal cell carcinoma. The novel p.55X nonsense mutation associated to bilateral RCC and retinal angioma in a 15-year-old male patient. CONCLUSION: We describe the earliest onset renal cell carcinoma in VHL disease reported so far in a 15-year-old boy with a nonsense VHL mutation. Individual tailoring of screening schedule based on molecular genetic status should be considered in order to diagnose serious complications as early as possible. Our observations add to the understanding of genotype-phenotype correlation in VHL disease and can be useful for genetic counseling and follow-up of VHL patients.

Epigallocatechin-3-gallate and penta-O-galloyl-ß-D-glucose inhibit protein phosphatase-1.

Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine/threonine residues in cells. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant δ-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC(50) values of 0.47-1.35 µm and 0.26-0.4 µm, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC(50) values of 15 and 6.6 µm, respectively. The structure-inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl/oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.