[Anthra-cycline-Induced Cardiotoxicity: the Role of Genetic Predictors].

Aim      To evaluate the predictive significance of gene polymorphism in endothelin-1 type 2A receptor, NADPH oxidase, p53 protein, endothelial nitric oxide synthase, caspase 8, interleukin-1ß, tumor necrosis factor-α, superoxide dismutase-2, glutathione peroxidase-1, ß1-adrenoceptor, angiotensin-converting enzyme, and matrix metalloproteinase-3 (MMP-3) genes in evaluating the risk of anthracycline-induced cardiotoxicity (AIC) in women without concurrent cardiovascular diseases (CVD).Material and methods  This study included 176 women aged 45.0 [42.0; 50.0] years with breast cancer without concurrent CVD who were scheduled for polychemotherapy (PCT) with anthracycline antibiotics. Echocardiography was performed for all patients at baseline and at 12 months after the end of PCT course. Genetic polymorphism was determined with the polymerase chain reaction.Results At 12 months, all patients were in remission of the underlying disease. They were retrospectively included into 2 groups: 1st group, 52 patients with AIC and 2nd group, 124 women without AIC symptoms. The development of AIC was associated with the presence of the p53 protein gene Arg / Arg genotype (odds ratio (OR), 2.972; p=0.001), NOS3 gene T / T genotype (OR, 3.059; p=0.018), NADPH oxidase gene T / T genotype (OR, 2.753; p=0.008), GPX1 gene C / C genotype (OR, 2.345; p=0.007), MMP-3 gene 5A / 5A genotype (OR, 2.753; p=0.008), and ADRB1 gene G / G genotype (OR, 3.271; p=0.043).Conclusion      Evaluation of genetic polymorphism in p53 protein (rs1042522), NOS3 (rs1799983), NADPH-oxidase (rs4673), GPX1 (rs1050450), ADRB1 (Arg389Gly, rs1801253), and MMP-3 (rs3025058) genes can be recommended for use prior to starting chemotherapy in women with breast cancer without CVD for assessing the risk of AIC. A maximum risk of cardiotoxicity is associated with the presence of the p53 protein gene Arg / Arg genotype and NOS3 gene T / T genotype.

Heart failure with preserved left ventricular ejection fraction in patients with obstructive sleep apnea syndrome: prognostic value of biomarkers.

Aim      To study the role of soluble ST2 (sST2), N-terminal pro-brain natriuretic peptide (NT-proBNP), and С-reactive protein (CRP) in patients with chronic heart failure and preserved left ventricular ejection fraction (CHF with pLVEF) and syndrome of obstructive sleep apnea (SOSA) in stratification of the risk for development of cardiovascular complications (CVC) during one month of a prospective observation.Material and methods  The study included 71 men with SOSA with an apnea/hypopnea index (AHI) >15 per hour, abdominal obesity, and arterial hypertension. Polysomnographic study and echocardiography according to a standard protocol with additional evaluation of left ventricular myocardial fractional changes and work index were performed for all patients at baseline and after 12 months of observation. Serum concentrations of sST2 , NT-proBNP, and CRP were measured at baseline by enzyme-linked immunoassay (ELISA).Results The ROC analysis showed that the cutoff point characterizing the development of CVC were sST2 concentrations ≥29.67 ng/l (area under the curve, AUC, 0.773, sensitivity 65.71 %, specificity 86.11 %; p<0.0001) while concentrations of NT-proBNP (AUC 0.619; p=0.081) and CRP (AUC 0.511; р=0.869) were not prognostic markers for the risk of CVC. According to data of the ROC analysis, all patients were divided into 2 groups based on the sST2 cutoff point: group 1 included 29 patients with ST2 ≥29.67 ng/l and group 2 included 42 patients with ST2 <29.67 ng/l. The Kaplan-Meyer analysis showed that the incidence of CVC was higher in group 1 than in group 2 (79.3 and 28.6 %, respectively, p<0.001). The regression analysis showed that adding values of AHI and left ventricular myocardial mass index (LVMMI) to sST2 in the model increased the analysis predictive significance.Conclusion      Measuring sST2 concentration may be used as a noninvasive marker for assessment of the risk of CVC development in patients with CHF with pLVEF and SOSA within 12 months of observation. Adding AHI and LVMMI values to the model increases the predictive significance of the analysis.

[Role of markers of inflammation and endothelial dysfunction in the prognosis of the development of cardiovascular complications in patients with coronary artery disease and metabolic syndrome after coronary stenting].

Aim To study the role of inflammation markers and endothelial dysfunction in predicting the risk of cardiovascular event following a percutaneous coronary intervention (PCI) in patients with ischemic heart disease (IHD) and metabolic syndrome (MS).Materials and methods 80 patients (72 men; median age, 56 (50;63) years) with IHD and PCI were evaluated. Based on the presence of MS according to NCEP-ATP III criteria, patients were divided into two groups, group 1 without MS (n=32) and group 2 with MS (n=48). The control age- and sex-matched group included 15 people without cardiovascular diseases. Serum concentrations of tumor necrosis factor α (TNFα), interleukin 6 (IL-6), IL-10, lipoprotein-associated phospholipase A2 (LP-PLA2), and endothelin 1 were measured by enzyme-linked immunosorbent assay (ELISA). Patients were followed up for 12 months after PCI with evaluation of the incidence of adverse cardiovascular events. Statistical analysis was performed with Statistica 10.0 and Medcalc 19.2.6 software. Differences between variables were considered statistically significant at р<0.05. Potential predictors were determined by the ROC analysis with construction of ROC curves, calculation of AUC (area under the curve), identification of COP (cut-off point by the Youden's index), and sensitivity (Se) and specificity corresponding to the COP.Results Patients with MS had statistically significantly higher serum levels of inflammatory markers than patients of the control group. Concentration of the intravascular inflammation marker, PL-PLA2, was 2.7 times higher in group 1 and 5.1 times higher in group 2 than in the control group (р<0.001). Concentrations of endothelin 1 were 1.9 times higher in group 1 and 3.7 times higher in the MS group compared to the control. At one year after PCI, the incidence of adverse outcomes in the form of cardiovascular events was higher for patients with MS: 10 (20.8 %) cases of stent restenosis and 13 (27.1 %) episodes of coronary atherosclerosis progression according to results of repeated coronarography vs. 2 (6.3%) restenosis cases (χ2-10.853; р=0.002) and 2 (6.3%) episodes of atherosclerosis progression (χ2-23.651; р=0.001) for patients without MS. The groups did not differ in rates of myocardial infarction and cardiac death. The most significant predictors of unfavorable prognosis were LP-PLA2 concentration >983.83 ng/ml (area under the ROC curve, 0.867; sensitivity, 80 %; specificity, 100%; р<0.001) and endothelin 1 overexpression >0.852 fmol/ml (area under the ROC curve, 0.885; sensitivity, 85.5 %; specificity, 83.6 %; р<0.001).Conclusion Patients with MS were characterized by more pronounced imbalance of pro- and anti-inflammatory factors. Concentrations of LP-PLA2 >983.83 ng/ml and endothelin 1 >0.852 fmol/ml were shown to be predictors of unfavorable prognosis for patients with IHD and MS after PCI with coronary stenting.

[Prognostic role of p53 gene polymorphism in risk assessment of anthracycline-induced cardiotoxicity].

AIMS: To study the prognostic significance of polymorphism of the p53 gene (polymorphism Arg72Pro exon 4, rs1042522) on the development of cardiotoxic remodeling of the left ventricle and heart failure. MATERIAL AND METHODS: A total of 176 women with breast cancer who received anthracycline antibiotics as part of polychemotherapeutic treatment regimens were examined. Based on the results of the survey, 12 months after the end of polychemotherapy, patients in the remission of the underlying disease were divided into 2 groups: patients with cardiotoxic remodeling (52 patients) and women with preserved heart function (124 patients). All patients before the start of the course of chemotherapy, in the dynamics of treatment with anthracyclines and after therapy with such were carried out the study of echocardiographic parameters. All the patients were taken genetic material, followed by typing alleles of the gene for the protein p53 (rs1042522). RESULTS: Analysis of echocardiographic parameters in patients 12 months after the completion of polychemotherapy in comparison with those before treatment showed a significant difference in the final systolic (33 mm [31; 35] and 28 mm [26; 31], p<0.00001) and terminal diastolic dimensions (51 mm [49; 54.5] and 44 mm [42; 48.5], p=0.0003), as well as a significant decrease in the left ventricular ejection fraction (54.5% [51.5; 58] and 65.5% [62; 70], p<0.00001) in the group of women with developed anthracycline cardiotoxicity. The presence of the Arg/Arg genotype was associated with the development of cardiotoxic myocardial damage during polychemotherapy (OR=3.86, 95% C.I.=1.45-10.26, p=0.005). The Pro/Pro genotype has proved to be a protective factor (OR=0.26, 95% C.I.=0.09-0.69, p=0.015). The conclusion. Predicting the cardiotoxicity of chemotherapy using the polymorphism of the p53 gene is an effective measure of early pre-symptom diagnosis of an increased risk of anthracyclineinduced cardiotoxicity.

Prognostic role of ST2 in patients with chronic heart failure of ischemic etiology and carbohydrate metabolism disorders.

AIM: To study the role of soluble ST2 (sST2) in patients with coronary artery disease (CAD) and chronic heart failure (CHF) associated with carbohydrate metabolism disorders (impaired glucose tolerance (IGT) and type 2 diabetes mellitus (DM) in risk stratification of adverse cardiovascular events (ACE) for 12 months of follow-up. MATERIALS AND METHODS: We enrolled 118 patients with CAD and CHF I-III FC (NYHA) with the ejection fraction of left ventricular of 60 [46; 64] % aged 62.5 [57; 68] years. Serum sST2 levels were measured by enzyme immunoassay. RESULTS: Depending on the presence of carbohydrate metabolism disorders (CMD), the patients were divided into 3 groups: group 1 (n=65) included patients without CDM, group 2 (n=30) included with IGT, and group 3 (n=23) included with type 2 DM. Serum levels of sST2 in patients with CMD were significantly (p=0.011) higher than in patients without CMD, but in subgroups of patients with IGT and type 2 DM, the concentrations of sST2 did not differ. In group 1 sST2 levels were 30.51 [26.38; 37.06] ng/ml, and in group 2 and 3 - 37.97 [33.18; 47.48] and 41.45 [35.27; 50.37] ng/ml, respectively. There were statistically significant differences in the rate of adverse ACE in relation to sST2 levels: in spite of CMD, in subgroups with biomarker overexpression adverse CCC occurred more often (p<0.01). According to the ROC analysis, the sST2 level of 33.14 ng/ml with the sensitivity of 73.3% and the specificity of 75.0% can be considered as a marker of ACE development within 12 months of follow-up (AUC=0.77, 95% CI 0.59-0.89, p=0.002). CONCLUSION: In patients with CHF of ischemic etiology, the prognostic significance of sST2 are established as a biomarker of ACE development. In patients with CDM, sST2 levels are significantly higher than in those without CDM that is associated with a higher rate of ACE within 12 months of follow-up.

[The effect of bisphosphonate therapy on reducing the risk of cardiovascular complications associated with chronic heart failure, type 2 diabetes and osteoporosis in postmenopausal women].

AIM: To study the effectiveness of oral alendronate and ibandronate bisphosphonates for the prevention of cardiovascular complications in postmenopausal women with type 2 diabetes mellitus (DM) and osteoporosis during a 12-month prospective observation. MATERIALS AND METHODS: The study included 86 women with osteoporosis, chronic heart failure (CHF) and type 2 diabetes: the 1st group (n=52) included patients who received basic therapy for heart failure; the 2nd group (n=34) included patients who, in addition to the basic therapy of heart failure, were prescribed alendronic and ibandronic acid preparations for the treatment of osteoporosis. In order to identify the possibility of associating the studied factors with the nature of the course of heart failure, the patients were divided according to the results of a one - year follow - up into two subgroups: subgroup A (n=49) - patients with a favorable course of the disease and subgroup B (n=37) - patients with an unfavorable course of pathology. RESULTS AND DISCUSSION: After 12 months, a significant decrease in the levels of cerebral natriuretic peptide precursor (NT-proBNP), tumor necrosis factor-α, and interleukin-1ß was found in the group of women treated with bisphosphonates compared to baseline. Significant associations of NT-proBNP levels (p=0.02) and the studied cytokines (p=0.01) with an unfavorable course of heart failure were revealed. A significant association of bisphosphonate therapy with a favorable course of heart failure (p=0.01) was also revealed. The probability of developing adverse cardiovascular events during the year in the treatment of heart failure with basic therapy drugs with additional therapy of osteoporosis with bisphosphonates is significantly (p=0.0025) lower than the treatment of patients with heart failure with only basic therapy and not taking bisphosphonates for the treatment of osteoporosis. CONCLUSION: In postmenopausal women with associated cardiovascular pathology (CHF, type 2 diabetes and osteoporosis), prophylactic therapy with oral alendronate and ibandronate oral bisphosphonates is effective, reduces the risk of progression of heart failure, inhibits inflammatory mediators, positively affects the combined endpoints of comorbid cardiovascular pathology.

[Role of ST2 biomarker for the evaluation of myocardial remodeling in patients with ischemic heart failure with preserved ejection fraction].

AIM: 1) To study the role of soluble ST2 (sST2) in evaluation of left ventricular (LV) myocardial remodeling and 2) to evaluate the predictive value of sST2 for development of adverse cardiovascular events (CVE) during 12 months following myocardial revascularization in patients with ischemic heart disease (IHD) and chronic heart failure (CHF) with preserved LV ejection fraction (EF). MATERIALS AND METHODS: The study included 55 patients (42 men) with IHD and NYHA FC I-III CHF with LV EF 63 [59; 65] % aged 65 [58; 69] who were scheduled for myocardial revascularization. Echocardiographic evaluation of myocardial stress and myocardial remodeling indexes was performed for all patients. Content of sST2 was measured using enzyme immunoassay. RESULTS: Group 1 included patients with sST2 overexpression (≥35 ng/ml) (n=26; sST2-43.75 ng/ml) and group 2 - patients with the sST2 expression.

[Pathogenetic and Prognostic Role of Growth Factors in the Development of Chronic Heart Failure].

AIM: To study the role of growth factors ((vascular endothelial growth factor (VEGF), platelet derived growth factor AB (PDGF-AB) and basic fibroblast growth factor (FGF-basic)) in the development and progression of chronic heart failure (CHF) in patients with ishcemic heart disease (IHD). MATERIALS AND METHODS: We included in this study 94 patients with CHF. The control group comprised 32 persons. Blood serum levels of growth factors were determined at baseline and after 12 months of observation by enzyme-linked immunosorbent assay. RESULTS: VEGF, PDGF-AB and FGF-basic play an important role in the pathogenesis and progression of heart failure in patients with IHD, determining the increased risk of adverse cardiovascular events in this pathology. Serum activity of growth factors characterizes the severity and course of CHF: with disease progression levels of VEGF and FGF-basic decrease and PDGF-AB concentration increases. Initial low level of VEGF expression regardless of the sex of the patient's sex, significantly low level of FGF-basic and significantly high PDGF-AB in men characterizes unfavorable course of CHF. CONCLUSION: A correlation has been established between blood serum levels of VEGF, PDGF-AB and FGF-basic and severity and course of CHF.

[Myocardial and Arterial Stiffness Important Determinant of NT-ProBNP at Development of Heart Failure in Survivors of Myocardial Infarction].

AIM: To study diagnostic value of myocardial-arterial stiffness (MAS) as a determinant of N-terminal pro-brain natriuretic peptide (NT-proBNP) expression in patients with chronic heart failure (CHF) with ischemic or postinfarction left ventricular (LV) dysfunction. MATERIAL AND METHODS: We analyzed 6 months prognosis of 54 patients (mean age 61.7+/-8.6 years) with II-III NYHA class CHF divided into 2 groups: (I, n=18) with class II CHF and preserved LV ejection fraction (EF) (55+/-10.4%), (II, n=36) with class III CHF and low LF EF (30.4+/-6.8%). MAS was measured by echocardiography as ratio of arterial elasticity (Ea) and end-systolic elasticity of LV myocardium (Es). Serum NT-proBNP was measured by immunoenzyme assay. RESULTS: During 6 months follow-up one group II patient with initial NT-proBNP level 2020 rg/ml died. NT-proBNP level in group I was significantly lower than in group II (313 and 647 rg/ml, respectively). Ea/Es ratio was significantly higher (p=0.001) in group II. Multifactorial analysis demonstrated moderate correlation of NT-proBNP with Ea/Es ratio (r=0.50, p=0.0001) and negative correlation with LVEF (r=-0.45, =0.003) among patients with II-III class CHF. CONCLUSION: As correlation between symptoms and severity of clinical manifestations of ischemic or postinfarction cardiac dysfunction at development of CHF was not high it appears rational to consider MAS estimated by Ea/Es ratio as independent predictor of cardiovascular complications. Sufficiently close correlation between NT-proBNP and Ea/Es ratio allows to improve stratification of risk and to assess objectively prognosis of the disease using easier obtainable parameter Ea/Es in cases when possibility to measure NT-proBNP is not available.

[Role of soluble Fas ligand in myocardial remodeling, severity and outcomes of chronic heart failure]. / Rol' rastvorimogo Fas-liganda v remodelirovanii miokarda, tyazhesti techeniya i iskhodakh khronicheskoi serdechnoi nedostatochnosti.

AIM: to reveal the specific features of Fas ligand-mediated ischemic myocardial remodeling and those of chronic heart failure (CHF) development during a 12-month prospective follow-up. SUBJECTS AND METHODS: A total of 94 patients with ischemic CHF were examined and divided into 3 groups according to NYHA Functional Class (FC): 1) FC II CHF in 35 patients; 2) FC III CHF in 31; 3) FC IV CHF in 28. According to the results of the 12-month follow-up, the patients were randomized into 2 groups: A) 49 patients with a favorable course of cardiovascular disease and B) 45 patients with its poor course. Serum soluble Fas ligand (sFas-L) levels were measured by enzyme immunoassay. RESULTS: In the patients with CHF, the baseline sFas-L levels substantially exceeded that in the control group by 3-6 times (p<0.01). In the men with the poor course of CHF, the baseline serum sFAS-L levels (85.94±4.14 pg/ml) were significantly higher than that in the favorable CHF group (107.33±5.13 pg/ml; р=0.0015). ROC analysis of the sensitivity and specificity of cardiovascular risk stratification according to sFAS-L levels revealed the high prognostic value of this marker - ROC-Area±S.E. was 0.75±0.05 (95% confidence interval, 0.60 to 0.81; p=0.0005). There was a statistically significant moderate correlation of left ventricular (LV) ejection fraction with sFAS-L concentrations and a moderate direct correlation between serum sFAS-L concentrations and LV remodeling parameters. CONCLUSION: The serum level of sFas-L determines the development of ischemic LV remodeling and the severity of CHF, by increasing in proportion to the degree of disease progression. The determination of serum sFas-L levels assists in objectively estimating the severity of apoptosis and may be an important prognostic test to assess the course of CHF in patients with coronary heart disease.

[Assessment of the role of matrix metalloproteinase-3 gene polymorphism in the development of chronic heart failure].

AIM: To study the impact of a polymorphic variant of the matrix metalloproteinase-3 (MMP-3) gene on the development and course of chronic heart failure (CHF) in patients with coronary heart disease. SUBJECTS AND METHODS: A total of 277 patients with New York Heart Association (NYHA) Functional Class (FC) II-IV CHF were examined. MMP-3 -1171 5A/6A genetic polymorphism was studied by polymerase chain reaction. A control group included 136 patients (mean age 53.6 ± 4.8 years) with no signs of cardiovascular diseases, as evidenced by the examination. RESULTS: The frequency of the 5A allele and the 5A/5A genotype of the 1171 5A/6A polymorphic locus in the MMP-3 gene proved to be higher in the patients with CHF than that in the control group. Thus, the variability of the 5A allele (odds ratio (OR), 1.39; 95% confidence interval (CI): 1.033 to 1.869; p = 0.03) and the 5A/5A genotype (OR, 2.15; 95% CI: 1.131 to 4.070; p = 0.02) was associated with increased risk for CHF. There were significant differences in the frequency of MMP-3 alleles and genotypes in relation to FC of CHF. The frequency of the 5A/5A genotype was substantially higher in the patients with NYHA FC IV CHF than that in those with NYHA FC II CHF (32.8% versus 15.2%; p = 0.039). The frequency of the 5A allele was significantly higher in the patients with NYHA FC IV CHF than that in those with NYHA FC II CHF (55.5% and 39.3%; respectively; p = 0.019). Thus, the carriage of the 5A allele and the 5A/5A genotype of the 1171 5A/6A polymorphic locus in the MMP-3 gene is a risk factor of severe CHF. CONCLUSION: The determination of MMP-3 -1171 5A/6A polymorphism may be recommended for the early prediction of a risk for the development and severe course of CHF.

[Comparative evaluation of the impact of four-week therapy with amlodipine and atenolol on quality of life and blood lipid composition in patients with coronary heart disease associated with metabolic syndrome].

AIM: To comparatively estimate the time course of changes in key metabolic parameters and quality of life (QL) in patients with coronary heart disease during 4-week therapy with atenolol and amlodipine. SUBJECTS AND METHODS: The 4-week randomized open-label trial included 60 patients with functional classes II-III stable angina pectoris on exertion associated with metabolic syndrome (all male patients aged 29 to 62 years (mean age 48.1 +/- 0.9 years)). Along with the traditional studies accepted in specialized cardiology practice, QL was assessed using the EORTC QLO CORE 30 questionnaire prior to treatment and on the last day of the trial. RESULTS: Four-week therapy with the individually adjusted dosages of atenolol (68.7 +/- 4.17 mg/day) or amlodipine (5.5 +/- 0.34 mg/ day) ensured comparable positive changes in the subjective assessment of QL. CONCLUSION: The positive changes in exercise tolerance that was considered to be an objective indicator for physical improvement in the treatment with amlodipine were more pronounced than those in that with atenolol. Therapy with amlodipine caused no change in blood lipid parameters while that with atenolol was associated with a 9.7% increase in blood triglyceride concentrations.

Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A crystal structure of the isomerase domain.

BACKGROUND: Glucosamine 6-phosphate synthase (GlmS) catalyses the first step in hexosamine metabolism, converting fructose-6P (6 phosphate) into glucosamine-6P using glutamine as a nitrogen source. GlmS is a bienzyme complex consisting of two domains that catalyse glutamine hydrolysis and sugar-phosphate isomerisation, respectively. Knowledge of the three-dimensional structure of GlmS is essential for understanding the general principles of catalysis by ketol isomerases and the mechanism of nitrogen transfer in glutamine amidotransferases. RESULTS: The crystal structure of the isomerase domain of the Escherichia coli GlmS with the reaction product, glucosamine-6P, has been determined at 1.57 A resolution. It is comprised of two topologically identical subdomains, each of which is dominated by a nucleotide-binding motif of a flavodoxin type. The catalytic site is assembled by dimerisation of the protein. CONCLUSIONS: The isomerase active site of GlmS seems to be the result of evolution through gene duplication and subsequent dimerisation. Isomerisation of fructose-6P is likely to involve the formation of a Schiff base with Lys603 of the enzyme, the ring-opening step catalysed by His504, and the proton transfer from C1 to C2 of the substrate effected by Glu488. The highly conserved C-terminal fragment of the chain may play a key role in substrate binding, catalysis and communication with the glutaminase domain. The corresponding sequence pattern DXPXXLAK[SC]VT (in single-letter amino-acid code, where X is any amino acid and letters in brackets indicate that either serine or cysteine may take this position) may be considered as a fingerprint of GlmS.

Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.

BACKGROUND: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS). RESULTS: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 A resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of alpha helices. CONCLUSIONS: The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own alpha-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.

Purification, crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from Thermus thermophilus.

High yields of inorganic pyrophosphatase from Thermus thermophilus HB8 have been purified to homogeneity using anion exchange and hydrophobic chromatography. Crystals suitable for X-ray analysis were obtained by vapour diffusion using ammonium sulphate as precipitant. They belong to the rhombohedral space group R32, with unit cell dimensions a = b = 110.3 A and c = 82.0 A, with one subunit per asymmetric unit. The crystals diffract to 2.0 A resolution.

Crystal structure of the aspartic proteinase from Rhizomucor miehei at 2.15 A resolution.

The crystal structure of the aspartic proteinase from Rhizomucor miehei (RMP, EC 3. 4. 23. 23) has been refined to 2.15 A resolution to a crystallographic R-value of 0.215 and an Rfree of 0.281. The root-mean-square (r.m.s.) error for the atomic coordinates estimated from a Luzzati plot is 0.2 A. The r.m.s. deviations for the bond distances and bond angles from ideality are 0.01 A and 1.7 degrees, respectively. RMP contains two domains that consist predominantly of beta-sheets. A large substrate-binding cleft is clearly visible between the two domains, and the two catalytic residues Asp38 and Asp237 are located in the middle of the cleft with a water molecule bridging the carboxyl groups of Asp38 and Asp237. Due to crystal packing, the C-terminal domain is more mobile than the N-terminal domain. Most of the aspartic proteinases (except renin) reach their maximum activity at acidic pH. We propose that the optimum pH of each aspartic proteinase is determined by the electrostatic potential at the active site, which, in turn, is determined by the positions and orientations of all the residues near the active site. RMP is the most glycosylated among the aspartic proteinases. The carbohydrate moieties are linked to Asn79 and Asn188. Asn79 is in the middle of a beta-strand and Asn188 is on a surface loop in contrast to the previous hypothesis proposed by Brown and Yada that they are both on surface beta-turns. RMP has a very high thermal stability. The high thermal stability is probably due to the high level of glycosylation. We propose that the highly flexible carbohydrates act as heat reservoirs to stabilize the conformation of RMP and therefore give the enzyme a high level of thermal stability. Three-dimensional structural and sequence alignments of RMP with other aspartic proteinases show that RMP is most structurally homologous to that of Mucor pusillus (MPP), and differs from other fungal enzymes as much as it does from the mammalian enzymes. This suggests that RMP and MPP diverged from the main stream of aspartic proteinases at an early stage of evolution. The present study adds a second member to this subfamily of aspartic proteinases.

Preliminary crystallographic study of cyclohexadienyl dehydratase from Pseudomonas aeruginosa.

Single crystals of cyclohexadienyl dehydratase from Pseudomonas aeruginosa have been obtained by vapour diffusion from ammonium sulphate solution (pH 6.0) at 4 degrees C. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2 with a = b = 105.5 A and c = 165.0 A. The asymmetric unit contains at least one dimeric protein molecule with M(r) = 72 kDa. The crystals diffract to 3 A resolution and are suitable for an X-ray analysis.

Channeling of ammonia in glucosamine-6-phosphate synthase.

Glucosamine-6-phosphate synthase catalyses the first and rate-limiting step in hexosamine metabolism, converting fructose 6-phosphate into glucosamine 6-phosphate in the presence of glutamine. The crystal structure of the Escherichia coli enzyme reveals the domain organisation of the homodimeric molecule. The 18 A hydrophobic channel sequestered from the solvent connects the glutaminase and isomerase active sites, and provides a means of ammonia transfer from glutamine to sugar phosphate. The C-terminal decapeptide sandwiched between the two domains plays a central role in the transfer. Based on the structure, a mechanism of enzyme action and self-regulation is proposed. It involves large domain movements triggered by substrate binding that lead to the formation of the channel.

Crystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli.

The glutamine amidohydrolase and fructose 6-phosphate binding domains of glucosamine-6-phosphate synthase from Escherichia coli have been overexpressed, purified and crystallized for X-ray diffraction analysis. The crystals of the glutamine amidohydrolase domain belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 70.4 A, b = 82.5 A, c = 86.1 A, with two molecules in the asymmetric unit, and diffract to 1.9 A resolution. The native Patterson indicated pseudo c-face centering of the unit cell. The fructose 6-phosphate binding domain was crystallized in the hexagonal space group P6(1) or P6(5) with cell dimensions a = b = 63.5 A, c = 334.3 A and with two molecules in the asymmetric unit. Diffraction data to 2.6 A resolution have been collected.

Preliminary X-ray study of naproxen esterase from Bacillus subtilis.

Single crystals of naproxen esterase from Bacillus subtilis have been obtained from PEG6000 solutions at pH 8.0 by liquid-liquid diffusion while applying a temperature gradient from 4 degrees C to room temperature over a period of four weeks. The crystals belong to the trigonal space group P3(1)21 or P3(2)21 with a = b = 47.59 A and c = 212.91 A. The asymmetric part of the unit cell contains one protein molecule with M(r) = 33,771. The crystals diffract to at least 3.0 A resolution and are suitable for an X-ray structure analysis.