The genetics of antiplatelet drug resistance.

Platelets have a central role in the development of arterial thrombosis and subsequent cardiovascular events. An appreciation of this complex process has made antiplatelet therapy the cornerstone of cardiovascular disease management. However, numerous patients will experience a recurrent atherothrombotic vascular event despite adequate antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. In this article, we reviewed the antiplatelet mechanisms of aspirin and clopidogrel and the possible role of different polymorphisms, which may affect the efficacy of antiplatelet therapy. Heterogeneity in the way patients respond to aspirin and clopidogrel may in part reflect variation in cyclooxygenase (COX)-1, COX-2, glycoprotein (GP) Ib alpha, GP Ia/IIa, GP IIb/IIIa, UGT1A6*2, P2Y(1), P2Y(12), CYP2C9, CYP3A4 and CYP3A5 genotypes.

Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

The localization of the reaction center polypeptides (L, M, and H) in the membranes of both the wild-type, strain 2.4.1, and the carotenoidless mutant, R-26, of Rhodopseudomonas sphaeroides was determined by using affinity-purified antibodies specific for these proteins. Binding of the antibodies to reaction center subunits in spheroplasts was visualized in the electron microscope by immunoferritin labeling. The H and M subunits were labeled at both the cytoplasmic and the periplasmic surfaces of the membrane, whereas the L subunit was labeled only at the periplasmic surface of the membrane. Thus, the reaction center is asymmetrically oriented in the membrane with at least two subunits (H and M) spanning the membrane.

Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

Gender differences in hemorheological parameters of coronary artery disease patients.

Plasma fibrinogen concentration, plasma and whole blood viscosity (WBV) are independent risk factors of coronary artery disease (CAD). Fibrinogen seems to be a relatively stronger risk factor for women than for men, but men are more endangered by higher hematocrit (Hct) and WBV than women are. We have previously reported that a theoretically optimal Hct value can be determined using Hct/WBV ratio in healthy subjects, hyperlipidemic and Raynaud's disease patients. Our aim was to examine whether Hct/WBV ratio is differently correlated with Hct in men and women with proven CAD. In a retrospective study we analysed the hemorheological data of 162 CAD outpatients (107 men and 55 women). Coronary angiography, echocardiography and impedance cardiography were performed. Hemorheological parameters (Hct, fibrinogen level, plasma viscosity, WBV), blood picture, serum lipid concentrations were determined and Hct/WBV ratio was calculated. Mean ages of male and female patients were similar (54.9 and 55.4 years, respectively), but men had significantly higher coronary angiography score than women. Mean left ventricular ejection fraction, stroke volume index and cardiac index showed no significant differences in men and women. Similarly, lipid concentrations, fibrinogen levels and plasma viscosities demonstrated no statistical differences. However, Hct, WBV and Hct/WBV ratios were significantly higher in male than in female patients (p < 0.00001; p < 0.00001 and p < 0.005, respectively). The most striking gender difference was found in the correlation between Hct/WBV ratio and cardiac index. Men older than 56 years showed negative, women positive correlation (r = -0.485, p = 0.01; r = 0.468, p = 0.006, respectively). This study demonstrates that Hct/WBV ratio as a rheological oxygen carrying capacity parameter is positively correlated with the cardiac index as it can be expected. However, the correlation is negative in elder men indicating an unhealthy relation between hemodynamic and hemorheologic parameters.

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides.

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfield's theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ(-)B and free energy and kinetic relations between Q(-)AQB and QAQ(-)B.

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: (Formula: see text). We found that the charge recombination pathway of D+QAQ(-)B proceeds via the intermediate state D+Q(-)AQB, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product kAD-times the fraction of reaction centers in the Q-AQB state) with the observed recombination rate, kobsD+----D. The kinetic measurements were used to obtain the pH dependence (6.1 smaller than or equal to pH smaller than or equal to 11.7) of the free energy difference between the states Q(-)AQB and QAQ(-)B. At low pH (less than 9) QAQ(-)B is stabilized relative to Q(-)AQB by 67 meV, whereas at high pH Q(-)AQB is energetically favored. Both Q(-)A and Q(-)B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q(-)B provides the driving force for the forward electron transfer.

Characterization of primary reactants in bacterial photosynthesis. II. Kinetic studies of the light-induced EPR signal (g = 2.0026) and the optical absorbance changes at cryogenic temperatures.

We have shown that the rise and decay kinetics of the light-induced EPR signal are identical to the kinetics of the optical changes at 80 degrees K. This identity provides independent evidence that the EPR signal is due to the oxidized primary electron donor which is bacteriochlorophyll. The EPR and optical changes could be described by a model photochemical reaction scheme that takes into account spin-lattice relaxation. The optical decay rate was found to be temperature independent between 1.5 and 80 degrees K and to obey approximately first order kinetics. These results are consistent with the hypothesis that the charge recombination occurs via tunneling through a potential barrier. The decay constants at these temperatures were found to be the same for different bacterial species and strains. No differences were found between purified reaction centers of R. spheroides R-26 and whole cells. Reaction centers treated with sodium dodecylsulfate or urea were still photochemically active but showed a markedly different kinetic behavior. The decay constant may, therefore, serve as a probe to investigate the molecular environment of the primary reactants.

Light-induced proton uptake by photosynthetic reaction centers from Rhodobacter sphaeroides R-26.1. II. Protonation of the state DQAQB2-.

Proton uptake associated with the two-electron reduction of QB was investigated in reaction centers (RCs) from Rhodobacter sphaeroides R-26.1 using pH-sensitive dyes. An uptake of two protons was observed at pH < or = 7.5, consistent with the formation of the dihydroquinone QBH2. At higher pH, the proton uptake decreased with an apparent pKa of approx. 8.5, i.e., to 1.5 H+/2 e- at pH 8.5. A molecular model is presented in which the apparent pKa is due to the protonation of either the carbonyl oxygen on QB or of an amino acid residue near QB (e.g., His-L190). Experimental evidence in favor of the protonation of the oxygen is discussed. The kinetics of the electron transfer from QA-QB- to QAQB2- and the associated proton uptake were compared at several pH values and temperatures. At pH 8.5 (21.5 degrees C) the rate constants for the proton uptake and electron transfer are the same within the precision of the measurement. At lower pH, the proton uptake rate constant is smaller than that for electron transfer. The difference between the rate constants is temperature dependent, i.e., it varies from 12 +/- 4% at 21.5 degrees C (pH 7.5) to 28 +/- 4% at 4.0 degrees C (pH 7.5). We show that the kinetics can be explained by a previously proposed model (Paddock, M. L., McPherson, P. H., Feher, G. and Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. USA 87, 6803-6807) in which the uptake of two protons by doubly reduced QB occurs sequentially, one concomitant with and the other after electron transfer.

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes determination of the equilibrium between Q(-)AQB and QAQ(-)B.

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q(A)-Q(B) and Q(A) Q(-B). A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of α = [Q(A)-Q(B)I/(IQ(A)-Q(Bl)+ [Q(A)Q(-B)I) = 0.065 +/- 0.005 (T= 21°C, pH 8).

Light-induced electrogenic events associated with proton uptake upon forming QB- in bacterial wild-type and mutant reaction centers.

Light-induced voltage changes (electrogenic events) were measured in wild-type and site-directed mutants of reaction centers (RCs) from Rhodobacter sphaeroides oriented in a lipid monolayer adsorbed to a Teflon film. A rapid increase in voltage associated with charge separation was followed by a slower increase attributed to proton transfer from solution to protonatable amino-acid residues in the vicinity of the QB site. In native reaction centers the proton-transfer voltage had a pH-dependent amplitude with two peaks at pH 4.5 and pH 9.7, respectively. In the Glu-L212-->Gln RCs the high-pH peak was absent, whereas in the Asp-L213-->Asn RCs the low-pH peak was absent and the high-pH peak was shifted to lower pH by about 1.3 pH units. The amplitudes of the electrogenic phases as a function of pH follow approximately the measured proton uptake from solution (P.H. McPherson, M.Y. Okamura, G. Feher, Biochim. Biophys. Acta, vol. 934, 1988, pp. 348-368) and are ascribed to proton transfer to amino acid residues upon QB- formation. The peak around pH 9.7 is ascribed to proton uptake predominantly by Glu-L212 and the peak around pH 4.5 to proton uptake predominantly by Asp-L213 or a residue strongly interacting with Asp-L213.

Proton and electron transfer in bacterial reaction centers.

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.

ENDOR studies of the intermediate electron acceptor radical anion I-. in Photosystem II reaction centers.

The EPR and ENDOR characteristics of the intermediate electron acceptor radical anion I-. in Photosystem II (PS II) are shown to be identical in membrane particles and in the D1D2 cytochrome b-559 complex (Nanba, O. and Satoh, K. (1987) Proc. Natl. Acad. Sci. USA 84, 109-112). These findings provide further evidence that the D1D2 complex is the reaction center of PS II and show that the pheophytin binding site is intact. A hydrogen bond between I-. and the protein (GLU D1-130) is postulated on the basis of D2O exchange experiments. The ENDOR data of I-. and of the pheophytin a radical anion in different organic solvents are compared and the observed differences are related to structural changes of the molecule on the basis of molecular orbital calculations (RHF-INDO/SP). The importance of the orientation of the vinyl group (attached to ring I) on electron transfer is discussed.

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides.

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.

Studies of crystal growth mechanisms of proteins by electron microscopy.

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.

Proton transfer pathways and mechanism in bacterial reaction centers.

The focus of this minireview is to discuss the state of knowledge of the pathways and rates of proton transfer in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Protons involved in the light driven catalytic reduction of a quinone molecule QB to quinol QBH2 travel from the aqueous solution through well defined proton transfer pathways to the oxygen atoms of the quinone. Three main topics are discussed: (1) the pathways for proton transfer involving the residues: His-H126, His-H128, Asp-L210, Asp-M17, Asp-L213, Ser-L223 and Glu-L212, which were determined by a variety of methods including the use of proton uptake inhibiting metal ions (e.g. Zn2+ and Cd2+); (2) the rate constants for proton transfer, obtained from a 'chemical rescue' study was determined to be 2 x 10(5) s(-1) and 2 x 10(4) s(-1) for the proton uptake to Glu-L212 and QB-*, respectively; (3) structural studies of altered proton transfer pathways in revertant RCs that lack the key amino acid Asp-L213 show a series of structural changes that propagate toward L213 potentially allowing Glu-H173 to participate in the proton transfer processes.

Postnatal remodeling of the leptomeningeal vascular network as assessed by intravital fluorescence video-microscopy in the rat.

An intriguing characteristic of the ontogenic development of the cerebral vasculature is the rapid differentiation of the neonatal leptomeningeal vascular plexus into the mature, adult network form. The physiological and cellular mechanisms of this cerebrovascular remodeling process are unclear. The objective of this work was to determine and correlate changes in vascular density, network pattern and flow velocity in leptomeningeal microvessels of the rat during postnatal development in vivo. To this end, microvascular diameter, segment length, and vascular density of reconstructed leptomeningeal networks were measured from video-recordings of the microcirculation visualized through a cranial window in 0-15-day-old Sprague-Dawley rats. The velocity of erythrocytes in the microvessels was measured by frame to frame tracking of fluorescently labeled red blood cells. We found that surface vascular density (total vessel length per area), node density and segment density (object per area) decreased significantly by the second week after birth. Anastomosing vascular polygons, characteristic to newborn networks, became less numerous and larger in diameter during the postnatal 2-week period, indicating progressive rarefaction of the networks. Vessel diameter and red cell velocity showed transient increases at 1.5 weeks. The velocity/diameter ratio (V/D), an index of wall shear rate, increased by the age of 1.5 weeks and remained unchanged afterwards. There was a negative correlation between V/D and diameter at 1 week; this relationship was reversed to a positive correlation at 2 weeks. We conclude that postnatal remodeling of the leptomeningeal vascular network is associated with rarefaction and an adaptation of vessel caliber to wall shear rate. These changes may contribute to arterio-venous differentiation and redistribution of blood flow from the superficial to the intracortical vasculature in the developing brain.

Erythrocyte flow heterogeneity in the cerebrocortical capillary network.

The heterogeneity of erythrocyte flow velocity and erythrocyte flux and their dependence on decreased cerebral perfusion pressure were studied in the rat cerebral cortex using intravital video microscopy. With decreased perfusion pressure, both mean and range of erythrocyte flow velocity of individual capillaries was reduced. Both cell velocity and cell flux decreased more in high flow capillaries than in low flow capillaries. The results are compatible with the hypothesis that redistribution of capillary flow during hypotension may help to maintain the perfusion of arterio-venous capillary flow pathways. Although the data are preliminary, they represent the first direct measurements of capillary flow path length and transit time in the capillary network of the cerebral cortex.

[Idiopathic priapism in childhood]. / Idiopathiás priapismus gyermekkorban.

The authors described the operation technique of the spongiocavernosum shunt in connection with a successfully operated patient. In their case the Quackels procedure was applied with minor modifications, since the conservative one wasn't successful. Their patient is perfectly healthy functionally too. The etiologic factors could not be clarified.