The Development and Study of Recombinant Immunoglobulin A to Hemagglutinins of the Influenza Virus.

We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.

[Streptokinase and Staphylokinase: Differences in the Kinetics and Mechanism of Their Interaction with Plasminogen, Inhibitors and Fibrin].

Comparative in vitro study of the kinetics of various reactions involved in the process of thrombolysis initiated by streptokinase (SK) and staphylokinase (STA) was carried out. It was shown that at the interaction of an equimolar ratio of plasminogen (Pg) with SK or STA the rate of formation and the specific esterase activity of the complex plasmin (Pm) · SK are higher than those of the complex Pm · STA. The catalytic efficiency (kcat/Km) of hydrolysis of the chromogenic plasmin substrates by Pm · SK complex was 2 times higher than by Pm · STA complex. In the absence of fibrin catalytic efficiency (kPg/K(Pg)) of activation of Glu-plasminogen and Lys-plasminogen glycoform II by Pm · SK complex was higher than by Pm · STA complex, but the pres- ence of fibrin increased kPg/K(Pg)) activation of both plasminogens by Pm · STA complex significantly stronger than by Pm · SK complex due to the decrease in K(Pg)). In contrast to STA (15.5 kDa), SK molecule (47 kDa) creates significant steric hindrances for the interaction of plasmin in Pm · SK complex with protein inhibi- tors. In addition, SK caused greater fibrinogen degradation than STA. It is shown that Pm · SK and Pm · STA complexes lyse fibrin clots in buffer with similar rates, while the rate of lysis of plasma clots, immersed in plas- ma, by Pm · STA complex are significantly higher than those by Pm · SK complex. It was revealed that the species specificity of STA and S K is determined mainly by the rate of formation and the efficiency of Pm · SK and Pm · STA complexes in the activation of autologous plasminogen. The lysis efficiency of plasma clots of mammals fell in the series: human > dog > rabbit for SK and the dog > human > rabbit for STA. The results show that in the purified system SK is a more effective activator of plasminogen than STA. In the system con- taining fibrin and α2-AP, the activator and fibrinolytic activities of STA are higher than those of SK, due to the increased stability in plasma and fibrin specificity of STA, the fast reaction of the complex Pm · STA with α2AP and the ability of the STA to recyclization in the presence of α2AP.

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system.

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1-3, K1-4, and K1-4.5 (0-2 microM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 microM) had no effect, while 2 microM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.

Mechanism of action of omega-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase.

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.

Properties of hexahistidine-tagged organophosphate hydrolase.

The catalytic properties of organophosphate hydrolase (OPH) containing a hexahistidine tag His6 (His6-OPH) and purified to 98% homogeneity were investigated. The pH optimum of enzymatic activity and isoelectric point of His6-OPH, which were shown to be 10.5 and 8.5, respectively, are shifted to the alkaline range as compared to the same parameters of the native OPH. The recombinant enzyme possessed improved catalytic activity towards S-containing substrates: the catalytic efficiency of methylparathion hydrolysis by His6-OPH is 4.2 x 10(6) M(-1) x sec(-1), whereas by native OPH it is 3.5 x 10(5) M(-1) x sec(-1).

Synergy of in vitro thrombolytic action of combinations of recombinant staphylokinase and single-chain urokinase-type plasminogen activator.

Kinetics of lysis of human plasma clots immersed in plasma were studied in vitro at 37 degrees C under the influence of recombinant staphylokinase, single-chain urokinase-type plasminogen activator (scu-PA), and their simultaneous and consecutive combinations. Staphylokinase and scu-PA caused concentration- and time-dependent lysis of the clots; 32 nM staphylokinase and 75 nM scu-PA separately caused 50% lysis in 4 h. At these equally effective concentrations staphylokinase in 4 h induced a significantly lesser exhaustion of the plasma plasminogen, alpha(2)-antiplasmin, and fibrinogen than scu-PA. Combinations of staphylokinase (<30 nM) and scu-PA (<75 nM) rendered synergic thrombolytic action on the clots. The synergy of thrombolytic action was more pronounced on the simultaneous addition of the two agents than on their consecutive addition, scu-PA 30 min after staphylokinase. In 4 h after the addition, staphylokinase (25 nM) or scu-PA (15 nM) induced 24% and 2% lysis, respectively, whereas the simultaneous and consecutive combination of the same concentrations of these agents induced 58% and 50% lysis, respectively. The simultaneous combination of 15 nM staphylokinase and 15 nM scu-PA resulted in maximal 3.8-fold increase in the thrombolytic effect as compared to the expected total effect of the individual agents. Synergic combinations of the two agents caused lesser exhaustion of plasma plasminogen, alpha(2)-antiplasmin, and fibrinogen as compared with the expected total effect of these agents used separately. Thus, simultaneous and consecutive combinations of staphylokinase and scu-PA in a relatively narrow range of their concentrations possessed synergistic fibrin-selective thrombolytic action on the plasma clot in vitro.

Direct and electrically wired bioelectrocatalysis by hydrogenase from Thiocapsa roseopersicina.

Hydrogen enzyme electrodes based on direct and mediated bioelectrocatalysis were developed. Direct bioelectrocatalysis of hydrogen oxidation/evolution was observed for hydrogenase adsorbed on carbon filament material. The equilibrium hydrogen potential was achieved on mediatorless hydrogen enzyme electrodes in hydrogen atmosphere. The electrocatalytic activity of hydrogenase in direct bioelectrocatalysis of hydrogen oxidation was two orders of magnitude higher compared to platinum. The reported electrode remained 50% activity after 6 months of storage with periodical testing. Wired bioelectrocatalysis was achieved by adsorption of hydrogenase onto electropolymerized redox mediator N-methyl-N'-(12-pyrrol-1-yl-dodecyl)-4,4'-bipyridinium ditetrafluoroborate.

Experimental and theoretical investigation of arachidonic acid uptake in macrophages.

The kinetics of 3H-labeled arachidonic acid (AA, 10(-10)-10(-5) M) incorporation into murine peritoneal macrophages was investigated. During the incorporation of AA into the cells, the steady state was reached at 10 h. The level of incorporation consisted of 48-50% for nanomolar concentrations and 28-30% for micromolar concentrations of AA. Exogenous AA in micromolar but not nanomolar concentrations stimulated [3H]AA release from intracellular stores of pre-labeled cells. A mathematical model fitting the behavior of the experimental system is proposed. The difference in the level of uptake of AA in nanomolar and micromolar concentrations is explained by the activation of AA release from intracellular stores at high concentrations of exogenous AA.

The hyperexponential growth of the human population on a macrohistorical scale.

A qualitative comparison of human population dynamics with some other biological populations growth in free regime is given in this paper along with a quantitative description of human population growth in the last 2000 years. It is shown that human population growth law is very different from the growth laws of other populations: all biological populations grow according to exponential law, whereas the human population grows according to hyperexponential law which essentially moves ahead of the exponential one. It is found that unlike cell populations, the differential equation of rate of human population growth is of second order. This phenomenon can be described due to accelerating function which grows simultaneously with human population number. An analytical hyperexponential equation, which allows to describe dynamics of human population, was obtained.

Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein.

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.

Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions.

The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp. has one of the longest fluorescence waves and one of the most complex absorbance spectra among the family of fluorescent proteins. In this work we found that with time DsRed fluorescence decreases under mildly acidic conditions (pH 4.0-4.8) in a pH-dependent manner, and this fluorescence inactivation could be partially recovered by subsequent re-alkalization. The DsRed absorbance and circular dichroism spectra under these conditions revealed that the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation determined that native DsRed formed a tight tetramer under various native conditions. Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and the inactivation of fluorescence can be caused by protonation of a single ionogenic group in each monomer of DsRed tetramer.

Anaerobic toxicity and biodegradability of hydrolysis products of chemical warfare agents.

The toxicity and biodegradability of the main hydrolysis products of chemical warfare agents were investigated under methanogenic conditions. Among the tested substances, only MPhA does not have any toxic effect with regard to the aceticlastic methanogenic activity. The toxicity of other compounds varied between moderate (TDG, mercaptoethanol) to strong (ethanolamine, diisobutyl ester of MPhA). Biodegradability tests showed that all the products of chemical detoxification of mustard gas (ethanolamine, ethylene glycol, TDG, mercaptoethanol) can be biomineralized under methanogenic conditions. On the contrary, phosphorus-containing compounds from the chemical detoxification of nerve warfare agents (Sarin, Soman, Vx-gases) are quite persistent under these conditions.

Arachidonic acid regulation of prostanoid synthesis in macrophages.

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.

Kinetic properties of the activator complexes plasmin--staphylokinase and plasmin(ogen)--streptokinase in vitro.

Comparative kinetic and electrophoretic study of the interaction of plasminogen (PG) with equimolar concentrations of staphylokinase (SPK) and streptokinase (SK) at 4 and 37 degreesC showed that the PG--SK complex has fibrinolytic and esterase activities, whereas the PG--SPK complex was inactive. Both esterase and fibrinolytic activities were enhanced during the conversion of the PG--SPK complex to the complex of plasmin (PL) with SPK (PL--SPK) at 37 and 4 degreesC, while the PG--SK complex was rapidly converted to the PL--SK complex with higher esterase activity only at 37 degreesC. The catalytic efficiency of Z-Lys-pNP hydrolysis (kcat/Km) by the preformed PL--SPK complex was twofold lower than that in the case of the PL--SK complex. Incubation of the PL--SPK and PG--SK(PL--SK) complexes at 37 degreesC for 24 h was associated with the degradation of the proteins and with different kinetics of lowering of esterase, plasminogen activator, and fibrinolytic activities. The PL--SPK complex was considerably more stable than the PG--SK(PL--SK) complex; streptokinase degraded more rapidly than staphylokinase. Kinetics of lysis of fibrin clots by the two complexes were similar, but the efficiency of lysis of plasma clots by the PL--SPK complex was significantly higher than that in the case of the PG--SK(PL--SK) complex (at 0.03-1 microM). Probably, unlike streptokinase, staphylokinase which is less susceptible to degradation in the PL--SPK complex and is released from the triple complex alpha2-antiplasmin--PL--SPK, forms a potentially highly active new complex with free molecules of plasminogen in the plasma.

Enzyme self-inactivation is a main limitation of the preparation of eicosanoids. Enzymatic synthesis of thromboxane and 12(S)-hydroxytetraenoic acid.

Synthesis of prostanoids is accompanied by various processes reducing the product yield. These processes are also known to affect syntheses of thromboxane (TX) and 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE). Partially purified preparations of TX synthase and prostaglandin (PG) synthase were used to optimize TX synthesis with respect to concentrations of the enzymes and eicosapolyenoic acid (EPA). Conditions for the maximum product yield and the minimum consumption of enzymes were determined. Consumption of the TX synthase was large owing to its inactivation during the reaction and the nonenzymatic destruction of the intermediate product PG-endoperoxide. Separate addition of PG and TX synthases increased the product yield by preventing EPA sorption on ballast proteins. Microsomal 12-lipoxygenase (12-LO) was also shown to be inactivated during the reaction, and this process was the major limitation of 12-HETE synthesis. Lipoxygenase reaction in the presence of some reducing agents led to a considerable increase of the 12-HETE yield, supposedly by preventing further oxidation of the 12-LO reaction product 12-hydroperoxy derivative of eicosatetraenoic acid. The possibility of using human blood platelet microsomes for preparation of some derivatives of EPAs is discussed.

Laccase of Coriolus zonatus: isolation, purification, and some physicochemical properties.

Laccase is one of the lignolytic enzymes found in liquid cultures of the fungus Coriolus zonatus in defined medium. The enzyme was isolated from culture liquid and characterized. Laccase from C. zonatus is a single-chain protein with a molecular mass of 60 kDa. Carbohydrate moiety of enzyme consisted of mannose, galactose and N-acetyl-glucosamine in a ratio of 6:2:0.6, respectively, and comprised 10% of the entire molecule. Isoelectric point was detected at pH 4.6. Laccase was found to have a pH optimum of 4.9 and temperature optimum of 55 degrees C. Substrate specificity studies were conducted with catechol, K-ferrocyanide, hydroquinone, and sinapinic acid as substrates. The highest efficiency of catalysis was observed with sinapic acid as the substrate. The kinetic constants kcat and Km of this reaction were 624 s-1 and 7 microM, respectively.

Kinetic behavior of a receptor-enzyme system: a model for drug addiction.

A theoretical kinetic model describing the behavior of a receptor-enzyme system during formation of drug addiction is considered in this article. The model assumes concomitant action of narcotic on at least two targets with opposite effects. Theoretical kinetic principles that the system must satisfy for the development of drug addiction are formulated. These kinetic principles are the slow inactivation of receptor-enzyme system and the divergence of characteristic times of dynamic concentration of product and enzyme.

Non-aqueous enzymology approach for improvement of reagentless mediator-based glucose biosensor.

The formation of Nafion membranes containing glucose oxidase and dimethylferrocene as a mediator was optimized using a previously reported non-aqueous enzymology approach for biosensor development. Enzyme immobilization in Nafion membranes was carried out from water-organic mixtures with a high content of organic solvent. The mediator based reagentless glucose electrode was tested in a flow injection system. The response towards glucose addition was stable: the reproducibility during 50 assays exceeded 95%. The response was linear over the glucose concentration range 0.5-50 mM.

Prostaglandin H synthase of mouse macrophages: inhibiting and activating action of ibuprofen.

The effect of low (10(-10)-10(-14 M) ibuprofen concentrations on the release of labeled arachidonic acid metabolites by mouse peritoneal macrophages containing a constitutive isoform of prostaglandin H synthase was investigated. It was found that during the activation the cells metabolized AA through the cyclooxygenase pathway, synthesizing PGE2 (110 +/- 10 ng per 10(6) cells), PGF(2 alpha) (120 +/- 15 ng per 10(6) cells), and TxB2 (48 +/- 5 ng per 10(6) cells). Incubation of the macrophages with 10(-12) M ibuprofen leads to a sharp increase of PGf2 and PGF(2 alpha) synthesis (315 +/- 84 and 320 +/- 20 ng per 10(6) cells, respectively). A microsomal preparation of macrophage PGH-synthase was compared with PGH-synthase of ram seminal vesicles and analogy of their properties was shown (Km = 5.3.10(-5) M, IC50, ibuprofen = 15 microM). Some particularities of the PGH-synthase consecutive isoform (PGH-synthase-1) cellular regulation are discussed.