EFFICACY AND SAFETY OF SINUS RHYTHM RESTORATION WITH AMIODARONE AND PROPAFENONE IN OUTPATIENTS, ECONOMIC ASPECTS (BASED ON DATA OF N.A. SEMASHKO NORTHERN MEDICAL CLINICAL CENTER)

Aim. To study efficacy and safey of sinus rhythm restoration with amiodarone and propafenone in outpatients with recurrent atrial fibrillation (AF), and to estimate pharmacoeconomic efficiency of such cardioversion in comparison with hospital treatment.Material and methods. Patients (n=199; aged 59.2±1.36) with paroxysmal (73.9%) or persistent (26.1%) AF were included into the multicenter prospective study. Follow-up period was 13.14 months (min 1 month, max 36 months). Patients with arrhythmia relapse <48 h received outpatient cardioversion with amiodarone or propafenone orally. Daily outpatient examination, including ECG and blood pressure monitoring every 30-60 min, was carried out to evaluate efficacy of cardioversion and hemodynamics. Phone contact with patients was available. Patients with unstable hemodynamics received cardioversion in hospital. Patients received propafenone in cumulative dose of 600 mg (150-300 mg every hour), and amiodarone 600-800 mg daily. The cost/effectiveness ratio (CER) was estimated. This ratio shows the cost of one unit of effect.Results. Outpatient cardioversion with amiodarone was started 24 h earlier (p=0.029) and with propafenone — 4.5 h earlier (p=0.002) than that in hospital. The average dose of amiodarone in ambulatory cardioversion was 1.3 times less (713.7±84,62 mg, p=0.345) than that in hospital cardioversion. Outpatient treatment with amiodarone restored sinus rhythm 8.7 h earlier, and with propafenone - 3.5 h earlier than the same treatments in hospital. Efficiency of hospital cardioversion with amiodarone was 70%, with propafenone - 80%, and efficiency of outpatient cardioversion — 96,1% and 98,4%, respectively. Outpatient treatment did not cause any severe side effects. Expenses for outpatient cardioversion made up 143 724.25 rubles, and for hospital cardioversion — 92 870.47 rubles. Average treatment costs for one patient in hospital was 6 times greater than for an outpatient. Outpatient cardioversion had the lowest CER (~1300 rubles%).Conclusion. Outpatient cardioversion with amiodarone or propafenone is not only effective and safe, but also economically feasible

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date

α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG‡0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG‡0 = 1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG‡F − ΔΔG‡H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, k cat/K m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxsid

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported - on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A. Database Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925. Enzymes Glycoside hydrolase family 7 reducing end acting cellobiohydrolase We report the characterization of the GH7 CBH secreted by ascomycete G. candidum (GcaCel7A). X-ray data revealed the first O-glycosylation in a GH7 crystal structure on a loop where the glycan influences loop contacts and interactions with the cellulose surface. Even though GcaCel7A structures indicate higher loop flexibility than H. jecorina Cel7A, molecular simulations suggest high processivity and low endo-initiation probability similar to HjeCel7A.</p