Negative Cooperativity in the Interaction of Prostaglandin H Synthase-1 with the Competitive Inhibitor Naproxen Can Be Described as the Interaction of a Non-competitive Inhibitor with Heterogeneous Enzyme Preparation.

The kinetic mechanism of the interaction of nonsteroidal anti-inflammatory drugs (NSAIDs) with their main pharmacological target, prostaglandin H synthase (PGHS), has not yet been established. We showed that inhibition of PGHS-1 from sheep vesicular glands by naproxen (a representative of NSAIDs) demonstrates a non-competitive character with respect to arachidonic acid and cannot be described within a framework of the commonly used kinetic schemes. However, it can be described by taking into account the negative cooperativity of naproxen binding to the cyclooxygenase active sites of the PGHS-1 homodimer (the first naproxen molecule forms a more stable complex (K1 = 0.1 µM) with the enzyme than the second naproxen molecule (K2 = 9.2 µM)). An apparent non-competitive interaction of PGHS-1 with naproxen is due to slow dissociation of the enzyme-inhibitor complexes. The same experimental data could also be described using commonly accepted kinetic schemes, assuming that naproxen interacts was a mixture of two enzyme species with the inhibition constants Kα = 0.05 µM and Kß = 18.3 µM. Theoretical analysis and numerical calculations show that the phenomenon of kinetic convergence of these two models has a general nature: when K2 >> K1, the kinetic patterns (for transient kinetics and equilibrium state) generated by the cooperative model could be described by a scheme assuming the presence of two enzyme forms with the inhibition constants Kα = K1/2, Kß = 2·K2. When K2 << K1, the cooperative model can be presented as a scheme with two inhibitor molecules simultaneously binding to the enzyme with the observed inhibition constant K (K = K1·K2). The assumption on the heterogeneity of the enzyme preparation in relation to its affinity to the inhibitor can be used instead of the assumption on the negative cooperativity of the enzyme-inhibitor interactions for convenient and easy practical description of such phenomena in enzymology, biotechnology, pharmacology, and other fields of science.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Inhibition of cyclooxygenase activity of prostaglandin-H-synthase by excess substrate (molecular oxygen).

For the cyclooxygenase reaction of prostaglandin-H-synthase isolated from ram vesicular glands, dependences of the initial reaction rate, the maximal yield of the product, and the rate constant of enzyme inactivation in the course of reaction on oxygen concentration were studied in the absence and in the presence of electron donor in the reaction medium. It is shown that in the absence of electron donor the cyclooxygenase reaction is strictly governed by Michaelis-Menten kinetics over a wide range of oxygen concentrations (5-800 µM). In the presence of electron donor in the reaction medium it was found that cyclooxygenase reaction is inhibited by an excess of dissolved oxygen: the maximal values of the initial reaction rate and yield of the product are attained at oxygen concentration 50 µM, and its increase to 500 µM causes twofold decrease in the initial rate and maximal yield. The rate constant of enzyme inactivation in the course of reaction increases on increase in oxygen concentration both in the presence and in the absence of electron donor.

Molecular oxygen (a substrate of the cyclooxygenase reaction) in the kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase.

Prostaglandin-H-synthase is a bifunctional enzyme catalyzing conversion of arachidonic acid into prostaglandin H2 as a result of cyclooxygenase and peroxidase reactions. The dependence of the rate of the cyclooxygenase reaction on oxygen concentration in the absence and in the presence of electron donor was determined. A two-dimensional kinetic scheme accounting for independent proceeding and mutual influence of the cyclooxygenase and peroxidase reactions and also for hierarchy of the rates of these reactions was used as a model. In the context of this model, it was shown that there are irreversible stages in the mechanism of the cyclooxygenase reaction between points of substrate donation (between donation of arachidonic acid and the first oxygen molecule and also between donation of two oxygen molecules).

Kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase. Effect of electron donors on the cyclooxygenase reaction.

Prostaglandin-H-synthase (PGHS, EC 1.14.99.1) catalyzes the first committed step in biosynthesis of all prostaglandins, thromboxanes, and prostacyclins by converting arachidonic acid to prostaglandin H(2) (PGH(2)). PGHS exhibits two enzymatic activities: cyclooxygenase activity converting arachidonic acid to prostaglandin G(2) (PGG(2)) and peroxidase activity reducing the hydroperoxide PGG(2) to the corresponding alcohol, PGH(2). Despite the many investigations of the kinetics of PGHS, many features such as the absence of competition and mutual activation between the cyclooxygenase and peroxidase activities cannot be explained in terms of existing schemes. In this work we have studied the influence of different electron donors (N,N,N ,N -tetramethyl-p-phenylenediamine, L-epinephrine, 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), potassium ferrocyanide) on the PGHS activities. The proposed scheme describes independent but interconnected cyclooxygenase and peroxidase activities of PGHS. It also explains the experimental data obtained in the present work and known from the literature.

Observation of a narrow charm-strange meson D(+)(sJ)(2632)-->D(+)(s)eta and D(0)K(+).

We report the first observation of a charm-strange meson D(+)(sJ)(2632) at a mass of 2632.5+/-1.7 MeV/c(2) in data from SELEX, the charm hadro-production experiment E781 at Fermilab. This state is seen in two decay modes, D(+)(s)eta and D0K+. In the D(+)(s)eta decay mode we observe a peak with 101 events over a combinatoric background of 54.9 events at a mass of 2635.4+/-3.3 MeV/c(2). There is a corresponding peak of 21 events over a background of 6.9 at 2631.5+/-2.0 MeV/c(2) in the decay mode D0K+. The decay width of this state is <17 MeV/c(2) at 90% confidence level. The relative branching ratio Gamma(D0K+)/Gamma(D(+)(s)eta) is 0.14+/-0.06. The mechanism that keeps this state narrow is unclear. Its decay pattern is also unusual, being dominated by the D(+)(s)eta decay mode.

First observation of the doubly charmed baryon Xi(+)(cc).

We observe a signal for the doubly charmed baryon Xi(+)(cc) in the charged decay mode Xi(+)(cc)-->Lambda(+)(c)K-pi(+) in data from SELEX, the charm hadroproduction experiment at Fermilab. We observe an excess of 15.9 events over an expected background of 6.1+/-0.5 events, a statistical significance of 6.3sigma. The observed mass of this state is 3519+/-1 MeV/c(2). The Gaussian mass width of this state is 3 MeV/c(2), consistent with resolution; its lifetime is less than 33 fs at 90% confidence.

Precision measurements of the lambda(+)(c) and D0 lifetimes.

We report new precision measurements of the lifetimes of the Lambda(+)(c) and D0 from SELEX, the charm hadroproduction experiment at Fermilab. Based upon 1630 Lambda(+)(c) and 10 210 D0 decays we observe lifetimes of tau[Lambda(+)(c)] = 198.1+/-7.0+/-5.6 fs and tau[D0] = 407.9+/-6.0+/-4.3 fs.

Observation of the Cabibbo-Suppressed Decay Ξ_{c}

We report the first observation of the Cabibbo-suppressed charm baryon decay Ξ_{c}^{+}→pK^{-}π^{+}. We observe 150±22±5 events for the signal. The data were accumulated using the SELEX spectrometer during the 1996-1997 fixed target run at Fermilab, chiefly from a 600 GeV/c Σ^{-} beam. The branching fractions of the decay relative to the Cabibbo-favored Ξ_{c}^{+}→Σ^{+}K^{-}π^{+} and Ξ_{c}^{+}→Ξ^{-}π^{+}π^{+} are measured to be B(Ξ_{c}^{+}→pK^{-}π^{+})/B(Ξ_{c}^{+}→Σ^{+}K^{-}π^{+})=0.22±0.06±0.03 and B(Ξ_{c}^{+}→pK^{-}π^{+})/B(Ξ_{c}^{+}→Ξ^{-}π^{+}π^{+})=0.20±0.04±0.02, respectively.

[The microbiocenosis of the large intestine in animals with constant radiation exposure in the area of the Chernobyl Atomic Electric Power Station]. / Mikrobiotsenoz tolstogo kishechnika zhivotnykh pri postoiannom radiatsionnom vozdeistvii v zone ChAES.

State of microbiocenosis of large intestine in rats and furo subjected to constant combined irradiation has been studied in full-scale experiments in the emergency rooms of the Chernobyl NPP and in the nearest zone of its effect. The higher diversity of microflora (as compared to initial one), sharp increase of the amount of Candida genus fungi, a decrease of the number of bifidobacteria and increase of the amount of conditionally pathogenic flora were observed in rats with the dose of external irradiation of 1.43 Gr absorbed for 14 days. The expressed associative character of disbioses is distinctly traced. In furo affected by the radion exceeding the background level feeding with products contaminated by radionuclides leads to changes whose individual character corresponds to the picture of disbiosis under radiation injury.

[Radionuclide transfer by migratory birds]. / Perenos radionuklidov pereletnymi ptitsami.

Evaluation of the zoogenic transfer of radionuclides from the 30-km zone around the Chernobyl NPP was necessary because of the enormous heavily polluted territory and mighty flow of migratory birds who tended to large rivers, the Dnieper and Pripyat. The integral estimate of the transferred amount was obtained as a product of three variables: the transfer factor (0.0077 m2/kg for 137Cs; 0.0107 m2/kg for 90Sr), the density of birds (0.002 kg/m2, at the mass of migrants about 5000 t per year), and the total fund of radionuclides throughout the territory. The upper estimated limit of the annual transfer rate was 5.5 Ci (2E + 11 Bq) for 137 Cs and 1.8 Ci (6.7E + 10 Bq) for 90 Sr. Restrictions of hunting are recommended within the northern part of the Kiev reservoir.

[Heterophilic granulocytes of rats during irradiation]. / Geterofil'nye granulotsity krys pri obluchenii.

The bone marrow eosinophils of rats subjected to irradiation are capable of secreting a peroxidase system obtained by neutrophils and endotheliocytes. As a result, there appear heterophil granulocytes in the peripheral blood.