Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.

Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri.

Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+). Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

Reconstitution of dimethylamine:coenzyme M methyl transfer with a discrete corrinoid protein and two methyltransferases purified from Methanosarcina barkeri.

Methyl transfer from dimethylamine to coenzyme M was reconstituted in vitro for the first time using only highly purified proteins. These proteins isolated from Methanosarcina barkeri included the previously unidentified corrinoid protein MtbC, which copurified with MtbA, the methylcorrinoid:Coenzyme M methyltransferase specific for methanogenesis from methylamines. MtbC binds 1.0 mol of corrinoid cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell extract. Purified MtbC and MtbA were used to assay and purify a dimethylamine:corrinoid methyltransferase, MtbB1. MtbB1 is a 230-kDa protein composed of 51-kDa subunits that do not possess a corrinoid prosthetic group. Purified MtbB1, MtbC, and MtbA were the sole protein requirements for in vitro dimethylamine:coenzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was optimal for coenzyme M methylation with dimethylamine. MtbB1 methylated either corrinoid bound to MtbC or free cob(I)alamin with dimethylamine, indicating MtbB1 carries an active site for dimethylamine demethylation and corrinoid methylation. Experiments in which different proteins of the resolved monomethylamine:coenzyme M methyl transfer reaction replaced proteins involved in dimethylamine:coenzyme M methyl transfer indicated high specificity of MtbB1 and MtbC in dimethylamine:coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to methylate coenzyme M during methanogenesis from dimethylamine.

Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa.

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.

[Effect of over-synthesis of cloned gene products on methanol metabolism in the recombinant methylotrophic yeast Hansenula polymorpha]. / Vliianie sverkhsinteza produktov klonirovannykh genov na metabolizm metanola u rekombinantnykh metilotrofnykh drozhzhei Hansenula polymorpha.

As is shown expression homologous (dihydroxyacetone kinase) and heterologous (HBsAg, beta-galactosidase) genes in methylotrophic yeasts Hansenula polymorpha DL1 negatively affects on the growth parameters of a host strain. The reducing of specific growth rate (mu max) and yield of biomass per the unit of a consumed substrate (Yx/s) were found in all recombinant strains grown on methanol. Overproduction of dihydroxyacetone kinase and beta-galactosidase in recombinant H. polymorpha was accompanied by two-fold increasing of the activity of alcohol oxidase, which is the first enzyme of methanol oxidation. Otherwise, the activity of formaldehyde dehydrogenase two-fold decreased in the recombinant strain overproducing HBsAg compared with the host strain. It is suggested that the over-synthesis of foreign proteins requiring an additional energetic and metabolic expenses might reduce the growth parameters and the activities of some enzymes of methanol metabolism in recombinant methylotrophic yeast H. polymorpha.

[Biosynthesis of somatotropin in a recombinant strain of Escherichia coli]. / Biosintez somatotropina rekombinantnym shtammom Escherichia coli.

A recombinant Escherichia coli K-12 strain was grown in the regime of chemostat with glucose limitation at a different flow rate and in the regime of turbidostat. The stability of its population and the dynamics of somatotropin biosynthesis were studied. The plasmid-containing strain became less stable as the flow rate in the fermenter dropped down, which was due, apparently, to a greater limitation. The level of somatotropin biosynthesis was higher at a low dilution rate (D = 0.075, 0.17 and 0.34 h-1). Possible factors responsible for this phenomenon are discussed.

[Population dynamics of Pseudomonas aeruginosa capable of assimilating p-xylene]. / Populiatsionnaia dinamika Pseudomonas aeruginosa, sposobnoi usvaivat' p-ksilol.

The paper describes the dynamics of clones in the population of Pseudomonas aeruginosa 2x, which appear spontaneously as the result of variability in the capacity to use aromatic compounds. A culture growing on p-xylene is most homogeneous, which may be attributed to the selective action of the medium. However, the greatest number of cells capable of growth on p-xylene is found in a culture grown in a medium containing glucose.

[Regulation of the enzyme activity of para-xylene catabolism in a spontaneous para-xylene-negative variant of Pseudomonas aeruginosa 2x79]. / Reguliatsiia aktivnosti fermentov katabolizma para-ksilola u spontannogo para-ksilolnegativnogo varianta Pseudomonas aeruginosa 2x79.

A spontaneous variant of Pseudomonas aeruginosa 2x unable to grow on p-xylene as the sole source of carbon and energy has been isolated. p-Xylenenegative variant of P. aeruginosa 2x79 differs from the wild type strain by the character of growth on the p-xylene oxidation intermediates p-toluate and protocatochuate. The cell of 2x79 variant inability to grow on p-xylene has been shown to be accompanied by the elimination of the activities of three enzymes - p-xylene methylhydroxylase, p-cresol methylhydroxylase and metapyrocatechase and by the considerable alteration in the regulation of orto-cleavage aromatic ring enzymes activity pyrocatechase and protocatechuate-3,4-dioxygenase. Possible reasons for appearing spontaneous variants 2x79 in the population of P. aeruginosa 2x growing on the p-xylene are being discussed.

[Characteristics of the key enzyme regulation of peripheral p-xylene metabolism in Pseudomonas aeruginosa]. / Osobennosti reguliatsii kliuchevykh fermentov periferiinogo metabolizma p-ksilola u Pseudomonas aeruginosa.

The regulation of p-xylene methylhydroxylase, metapyrocatechase, pyrocatechase and protocatechoate-3,4-dioxygenase was studied in Pseudomonas aeruginosa 2x. Methylhydroxylase, the first enzyme of p-xylene oxidation, was shown to be synthesized in the strain in a constitutive manner and to be regulated at the level of the enzyme activity. Metapyrocatechase, protocatechase and pyrocatechase are inducible enzymes; these are repressed to a different extent by the end products of p-xylene oxidation. Metapyrocatechase has a broader substrate specificity as compared to pyrocatechase and is induced by a greater number of substrates, the affinity for different substrates depending on the structure of an inductor. Presumably, two isofunctional metapyrocatechases exist in P. aeruginosa 2 x.