Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype

In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene. Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the k(cat)/K(m) value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1 x 10(7) M(-1)s(-1) and 9 x 10(5) M(-1)s(-1). Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM; K(d) = 65.3 nM vs 2.1 nM, in FIIa-MT67 and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the absence of TM was normal. The Arg67His mutation had a dramatic effect on the cleavage of protease-activated G protein-coupled receptor 1 (PAR-1) 38-60 peptide (k(cat/)K(m) = 4 x 10(7) M(-1)s(-1) to 1.2 x 10(6) M(-1)s(-1)). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second-order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulfate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the Arg67His mutation was associated with a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the Arg67His substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.     

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K(m) values of ≤ 5 10(4) M(-1)s(-1)). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k(cat)/K(m), and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K(m) values up to 0.6 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of ≤ 10(7) almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.     

Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-) to FAD and form H(2)O(2) as a product. Limiting rate constants of k(cat)K(M)(O(2)) = (5.7 +/- 1.8) x 10(2) M(-1).s(-1) and k(cat)K(M)(O(2)) = (1.5 +/- 0.3) x 10(6) M(-1).s(-1) are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O(2). Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal.mol(-1) (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal.mol(-1) (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal.mol(-1) (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution.     

Water dynamics and salt-activation of enzymes in organic media: mechanistic implications revealed by NMR spectroscopy

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg co-lyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D(2)O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, (tau(c))(D(2)O), in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (k(cat)/K(M))(app) of the biocatalyst preparation increased from 0.092 s(-1) x M(-1) (salt-free) to 1,140 s(-1) x M(-1) (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (k(cat)/K(M))(app) of 0.140 s(-1) x M(-1) (salt-free) to 12.8 s(-1) x M(-1) (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(k(cat)/K(M))(app)] and (tau(c))(D(2)O) were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (k(cat)/K(M))(app) and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzyme-bound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.     

Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding

The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)(25)E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3Atriangle up1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (k(cat)/K(m)). Mutants H752A, H756A, and E825A had k(cat) of less than 0.0008 s(-1) to 0.0475 s(-1) compared to PDE3Atriangle up1, with 1.86 second(-1), with unchanged K(m). Although E866A had a k(cat) of 0.235 s(-1), the K(m) for cyclic adenosine monophosphate (cAMP) was increased 11-fold and the K(i) for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3Atriangle up1. The K(i) of H836A for cGMP was 177-fold higher than that of PDE3Atriangle up1. The K(m) for E971A was 5-fold higher than PDE3Atriangle up1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn(2+), Mg(2+), or Co(2+). This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.     

Characterization of the mutations in three patients with pyruvate dehydrogenase E1α deficiency

Summary The human pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. Defects in several of the seven subunits have been reported, but the majority of mutations affect the E1 component and especially the E1 subunit. However, the clinical presentation of patients with pyruvate dehydrogenase E1 deficiency is extremely variable. Dependency of the brain on pyruvate dehydrogenase activity and localization of the gene for the somatic form of the pyruvate dehydrogenase E1 subunit to the X chromosome provide the basis for a better understanding of the variation in the clinical manifestations. Further understanding of the function and interaction of subunits and the pathophysiology of pyruvate dehydrogenase deficiency necessitates the characterization of mutations in the pyruvate dehydrogenase complex. We report the analysis of three patients with pyruvate dehydrogenase E1 deficiency. One female has a three base pair deletion which affects dephosphorylation of the subunit. Of two males analysed, one has a two base pair deletion causing a shift in the reading frame. The other has a base change, resulting in an Arg to His substitution. All three mutations are located near the carboxyl terminus of the subunit.     

Mechanism of Action of 1-β-D-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole), A New Broad-Spectrum Antiviral Agent

The antiviral activity of the synthetic nucleoside, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), against measles virus in Vero cell cultures was substantially reversed by xanthosine, guanosine, and to a slightly lesser extent by inosine. Virazole 5'-phosphate was subsequently found to be a potent competitive inhibitor of inosine 5'-phosphate dehydrogenase (IMP:NAD(+) oxidoreductase, EC 1.2.1.14) isolated from Escherichia coli (K(m) = 1.8 x 10(-5) M) with a K(i) of 2.7 x 10(-7) M. Guanosine 5'-phosphate (GMP) was a competitive inhibitor of this enzyme with a K(i) of 7.7 x 10(-5) M. Virazole 5'-phosphate was similarly active against IMP dehydrogenase isolated from Ehrlich ascites tumor cells, with a K(i) of 2.5 x 10(-7) M. The K(m) for this enzyme was 1.8 x 10(-5) M, and the K(i) for GMP was 2.2 x 10(-4) M. These results suggest that the antiviral activity of Virazole might be due to the inhibition of GMP biosynthesis in the infected cell at the step involving the conversion of IMP to xanthosine 5'-phosphate. This inhibition would consequently result in inhibition of the synthesis of vital viral nucleic acid.     

Colossal kinetic isotope effects in proton-coupled electron transfer

The kinetics of reduction of benzoquinone (Q) to hydroquinone (H(2)Q) by the Os(IV) hydrazido (trans-[Os(IV)(tpy)(Cl)(2)(N(H)N(CH(2))(4)O)]-PF(6) = [1]PF(6), tpy = 2,2':6',2"-terpyridine), sulfilimido (trans-[Os(IV)-(tpy)(Cl)(2)(NS(H)-4-C(6)H(4)Me)]PF(6) = [2]PF(6)), and phosphoraniminato (trans-[Os(IV)(Tp)(Cl)(2)(NP(H)(Et)(2))] = [3], Tp(-) = tris(pyrazolyl)-borate) complexes have been studied in 1:1 (vol/vol) CH(3)CN/H(2)O and CH(3)CN/D(2)O (1.0 M in NH(4)PF(6)/KNO(3) at 25.0 +/- 0.1 degrees C). The reactions are first order in both [Q] and Os(IV) complex and occur by parallel pH-independent (k(1)) and pH-dependent (k(2)) pathways that can be separated by pH-dependent measurements. Saturation kinetics are observed for the acid-independent pathway, consistent with formation of a H-bonded intermediate (K(A)) followed by a redox step (k(red)). For the pH-independent pathway, k(1)(H(2)O)/k(1)(D(2)O) kinetic isotope effects are 455 +/- 8 for [1(+)], 198 +/- 6 for [2(+)], and 178 +/- 5 for [3]. These results provide an example of colossal kinetic isotope effects for proton-coupled electron transfer reactions involving nitrogen, sulfur, and phosphorus as proton-donor atoms.     

Type 10 17beta-hydroxysteroid dehydrogenase catalyzing the oxidation of steroid modulators of gamma-aminobutyric acid type A receptors

The steroids allopregnanolone and allotetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) are positive allosteric modulators of GABA(A) receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) and 5alpha-DHDOC, respectively, under the catalysis of human type 3 3alpha-hydroxysteroid dehydrogenase (HSD). However, brain enzymes catalyzing the conversion of such tetrahydrosteroids back to the corresponding 5alpha-dihydrosteroids remain to be identified. Characterization of human type 10 17beta-HSD provides a new insight into its importance for the oxidation of steroid modulators of GABA(A) receptors. The apparent catalytic efficiency (k(cat)/K(m)) of this enzyme for the oxidation of allopregnanolone and 3alpha,5alpha-THDOC are 432 and 1381 min(-1) mM(-1), respectively. This enzyme has negligible 3-ketosteroid reductase activity for 5alpha-DHP and 5alpha-DHDOC even in an acidic environment. Immunoreactivity against 17beta-HSD10 was found in a number of neuronal populations. Taken together, evidence suggests that 17beta-HSD10 is the brain enzyme capable of catalyzing the oxidation of steroid modulators of GABA(A) receptors.     

Human liver class I alcohol dehydrogenase gammagamma isozyme: the sole cytosolic 3beta-hydroxysteroid dehydrogenase of iso bile acids

3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.     

Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis

Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.     

Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis

The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg(2+)-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a k(cat) of 6.2 +/- 0.5 x 10(-3) s(-1) and a K(m) for farnesyl diphosphate of 62 +/- 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k(cat) of 3.2 +/- 0.4 x 10(-3) s(-1) and a twofold greater k(m) of 115 +/- 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.     

Reduction of Ferricytochrome c by Dithionite Ion: Electron Transfer by Parallel Adjacent and Remote Pathways

The kinetics of the reduction of horseheart ferricytochrome c by sodium dithionite (phosphate buffer-sodium chloride; pH 6.5, mu = 1.0, 25 degrees ) features two reaction pathways; one with the rate constant k(3) = 1.17 x 10(4) M(-1) sec(-1), the other with the rate constant k(1)k(2)/k(-1) = 6.0 x 10(4) M(-1) sec(-1). These pathways are interpreted in terms of remote attack (possibly by way of the exposed edge of the porphyrin system) and adjacent attack (requiring the opening of the heme crevice). The limiting rate for the adjacent pathway (k(1) = 30 sec(-1)) is in good agreement with the rate of heme-crevice opening of ferricytochrome c determined in other studies. The implication of the adjacent attack pathway to the function of cytochrome c in vivo is discussed.     

Fatal lactic acidosis due to deficiency of E1 component of the pyruvate dehydrogenase complex

Summary Pyruvate dehydrogenase complex deficiency is thought to be a common cause of lactic acidosis. We report a patient with lactic acidosis and intermittent weakness. The rate of oxidation of pyruvate by intact skeletal muscle and liver mitochondrial fractions was impaired and pyruvate dehydrogenase complex (PDC) activity was low. The amounts of immunoreactive dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase in liver and skeletal muscle mitochondrial fractions from the patient were normal. However, there were markedly lower concentrations of both the and subunits of the E1 component of PDC.     

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.     

Correlation between serum lactate dehydrogenase and stem cell mobilization

After observing a correlation between elevated serum lactate dehydrogenase (LDH) levels and good stem cell collections, retrospective data from 540 donors undergoing 650 stem cell apheresis procedures (87% autologous, 13% allogeneic) were studied to determine the correlation between preapheresis LDH levels and the absolute peripheral blood CD34+ cell count (PBCD34). PBCD34 (1-1611/mul; median 40) correlated modestly with leukocytes (0.5-118.2 x 10(9)/l; median 30.2) (r=0.16; P=0.00005) and poorly with platelets (16-660 x 10(9)/l; median 131) (r=0.02; P=0.69). The correlation between LDH (64-1664 IU/l; median 310) and PBCD34 was very strong (r=0.54; P<10(-48)). In multivariate regression analysis, serum LDH was the only factor correlating significantly with PBCD34. The correlation between serum LDH and PBCD34 was strong on the first day of collection (n=517; r=0.53; P<10(-37)), weakened on the second day (n=74; r=0.37; P=0.0009) and disappeared beyond day 2 (n=59; r=0.09; P=0.49). PBCD34 was significantly higher (median 53 versus median 11; P<0.00001) when LDH was elevated (n=511) compared to when LDH was normal (n=139). The relationship between serum LDH and PBCD34 was strong for autologous (r=0.54) as well as for allogeneic (r=0.41) collections. Our data suggest that it is reasonable to assume good stem cell mobilization and start apheresis if the LDH is elevated.     

支气管哮喘患者诱导痰炎性指标与气道反应性的关系

目的分析诱导痰中硝酸盐/亚硝酸盐(NO_3~-/NO_2~-)浓度和嗜酸粒细胞(EOS)计数与气道反应性的相关性,探讨上述指标对评估病情、调整支气管哮喘(简称哮喘)治疗方案的指导意义。方法 2003年2月至2004年6月华西医院哮喘门诊收集轻至中度非急性发作期哮喘患者35例,其中轻度9例,中度26例;经吸入糖皮质激素(ICS)和长效β_2受体激动剂(LABA)联合治疗1年,随访期间记录哮喘症状积分,测定气道反应性[以比气道传导率下降35%所需的激发剂浓度(PC_(35)sGaw)表示]、诱导痰中 EOS 计数和 NO_3~-/NO_2~-浓度。15名健康志愿者作为对照组,测定其诱导痰 EOS,NO_3~-/NO_2~-浓度。结果 35例患者中26例完成1年或以上的治疗和随访。26例患者 PC_(35)sGaw 治疗前为0.08g/L,治疗3个月为1.40g/L,随后7个月维持在2.64 g/L 水平。在治疗第3个月时,诱导痰NO_3~-/NO_2~-水平从治疗前的(734±72)×10~(-3)g/L 下降至(230±41)×10~(-3)g/L,差异有统计学意义(q=6.26,P<0.05),治疗7个月时 NO_3~-/NO_2~-水平降至(137±27)×10~(-3)g/L,与健康对照[(136±20)×10~(-3)g/L]比较差异无统计学意义(q=3.77,P>0.05)。治疗3个月后 EOS 计数为0.014±0.007,与健康对照(0.016±0.008)比较差异无统计学意义(q=2.94,P>0.05);随访期间任一时点PC_(35)sGaw 与 EOS 计数无相关(r_1=0.237,r_2=0.536,r_3=0.675,P 均>0.05)。5个月内 PC_(35)sGaw 与NO_3~-/NO_2~-水平呈负相关(r_1=-0.872,r_2=-0.653,r_3=-0.639,r_4=-0.656,P 均<0.05)。结论 PC_(35)sGaw 与诱导痰 NO_3~-/NO_2~-是反映哮喘气道炎症较为敏感的指标,可作为评价疗效及调整治疗方案的指标。     

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of k(cat) and k(cat)K(m) were 4.5 +/- 0.1 s(-1) and 0.49 +/- 0.1 x 10(6) M(-1).s(-1) in the absence of divalent metal ions, 14.7 +/- 2.2 s(-1) and 25.1 +/- 7.4 x 10(6) M(-1).s(-1) in the presence of Mg(2+) or Mn(2+), and 24.2 +/- 3.6 s(-1) and 2.4 +/- 0.7 x 10(6) M(-1).s(-1) when supported by VO(2+) or bis(acetylacetonato)oxovanadium(IV). Binding of VO(2+) to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg(2+). Electron paramagnetic resonance spectra of the ternary enzyme-VO(2+)-chelate-dUDP complex revealed a pattern of (31)P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO(2+) ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO(2+). We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO(2+) and the VO(2+)-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.     

Association of muscle glycogen synthase polymorphism with insulin resistance in type 2 diabetic patients

The aim of the present study is to investigate whether Met416Val (M416V) polymorphism of glycogen synthase (GYS1) gene is associated with insulin resistance in type 2 diabetes. In 100 type 2 diabetic subjects (66 men and 34 women), the M416V polymorphism of GYS1 gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as previously reported, and insulin resistance was assessed by euglycemic hyperinsulinemic clamp represented as M/I value, the mean of glucose infusion rate (M value) adjusted by steady state plasma insulin level. The means of age and body mass index (BMI) of the subjects were 53.1+/-11.6 (SD) years and 23.3+/-3.5 kg/m2. The allele frequencies of M416V polymorphism were 82.0% for MM, 16.0% for MV, and 2.0% for VV, and subjects were subsequently divided into V(+) group (n=18) and V(-) group (n=82) according to the presence or absence of V allele. There were no significant differences in age, BMI, blood pressure, fasting plasma glucose or insulin levels or glycosylated hemoglobin (HbA1c) levels between the V(+) and V(-) groups. No significant differences in either M or M/I value were found between the V(+) and V(-) groups (M value, 5.06+/-2.20 v 5.12+/-2.04 mg x kg(-1) x min(-1), P=.841; M/I value, 5.24+/-3.07 v 5.39+/-2.87 mg x kg(-1) x min(-1) x mU(-1) x L, P=.576). BMI showed the strongest independent contribution to M/I value, but the presence of V allele did not in multiple regression analysis. In conclusion, the M416V polymorphism of GYS1 gene is not associated with insulin resistance in type 2 diabetes.     

Neurohypophysial hormone receptors and second messengers in trout hepatocytes

Neurohypophysial hormone receptors and second messengers were studied in trout (Oncorhynchus mykiss) hepatocytes. Arginine vasotocin (AVT) and isotocin (IT) elicited a concentration-dependent inhibition of cAMP accumulation in the presence of 5x10(-8) M glucagon (maximal effect for 4.5x10(-7) M and 1.4x10(-7) M, half-maximal effect for 2.1x10(-8) M and 0.7x10(-8) M, AVT and IT respectively). The effect of glucagon was inhibited up to 90% by AVT and 80% by IT. While AVT inhibited (up to 50%) the basal cAMP production, IT had no such action. Specific V(1) or V(2) analogues (with reference to vasopressin in mammals) were used for pharmacological characterization of the type of neurohypophysial hormone receptor involved in this inhibition. The V(1) agonist [Phe(2), Orn(8)]-oxytocin inhibited the glucagon-stimulated cAMP production with a maximal effect for 6x10(-7) M and a half-maximal effect for 0.9x10(-8) M concentrations of the analogue. While the V(1) agonist reduced the glucagon-stimulated cAMP level by 70%, it showed only a tendency to reduce the basal level. The V(2) agonist [deamino(1), Val(4),d -Arg(8)]-vasopressin had no effect either on basal or on glucagon-stimulated cAMP production. The V(1) antagonist [d(CH(2))(5)(1), O-Me-Tyr(2), Arg(8)]-vasopressin totally reversed the 10(-8) M AVT-induced inhibition of 5x10(-8) M glucagon-stimulated cAMP production, whereas the V(2) antagonist [d(CH(2))(5)(1),d -Ile(2), Ile(4), Arg(8), Ala(9)]-vasopressin had no such effect. In this particular case, maximal and half-maximal effects of the V(1) antagonist were obtained for 2.3x10(-6) M and 1. 2x10(-6 )M respectively. Changes in intracellular calcium content were measured using the fluorescent probe FURA-2/AM. AVT and IT elicited a concentration-dependent increase in Ca(2+) accumulation. The comparison of the effect of 10(-8) M agonists versus AVT showed the following order of potency: AVT=IT>V(1) agonist>V(2) agonist. The V(1) antagonist reversed the AVT-induced Ca(2+) accumulation whereas the V(2) antagonist had no such effect. These results are taken as evidence for the presence in trout hepatocytes of neurohypophysial hormone receptors functionally close to the V(1a)-type linked to cAMP production and Ca(2+) mobilization.