Development of new analytical systems based on biocatalysers

A new amperometric analytical system based on biocatalysers is described. The study examined the use of immobilized multienzyme systems, sensitivity increase of devices by chemical amplification, the use of dehydrogenases, hydrolases and microorganisms and the application of biochemical cells as analytical devices. The macrokinetic dependence of the action of systems is considered and the means of their possible development are proposed.     

Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.     

Chemiluminescent immunoassay (CLIA) for the detection of brucellosis and tularaemia antigens

The detection of brucellosis and tularaemia infection agents is of particular interest for medical practice. The possibility of using enhanced chemiluminescence reactions for the determination of these agents is studied in this work. Light intensity depends on both the conjugate concentration used and the conditions at which the adsorption was performed. Optimal conditions for these test-systems were: ～ 20 μg/mL of Ig and 200 μg/mL (titre 1:20) of conjugate. As is seen from the chemiluminescent and spectrophotometric results the lowest determined concentrations are 10 and 30 ng/mL (for brucellosis) and 1 and 5 ng/mL (for tularaemia), respectively. Calibration curves in the antigen concentrations ranging from 10 to 2500 ng/mL (for brucellosis) and from 1 to 500 ng/mL (for tularaemia) are observed. Optical density depends linearly on the logarithm of the antigen concentration from 30 to 5000 ng/mL (for brucellosis) and from 5 to 250 ng/mL (for tularaemia). The results obtained permit the conclusion that the chemiluminescence method can be used in enzyme immunoanalysis for brucellosis and tularaemia antigens.     

Gene Polymorphisms of Micrornas in Helicobacter pylori-Induced High Risk Atrophic Gastritis and Gastric Cancer

Background and aims

MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population.

Methods

Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR.

Results

Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR −2.34, 95% CI 1.08–5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14–5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC.

Conclusions

Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.     

A 'branched' mechanism of the reverse reaction of yeast glutathione reductase. An estimation of the enzyme standard potential values from the steady-state kinetics data

The reduced glutathione-linked NADP+ reduction, catalyzed by yeast glutathione reductase, follows a 'sequential' or 'ping-pong' mechanism at high or low NADP+ concentrations, respectively. The pattern of the NADPH and NADP+ cross-inhibition reflects not only the competition for the binding site, but the shift of the reaction equilibrium as well. A 'branched' scheme of the glutathione reductase reaction is presented. The enzyme standard potential (-255 mV, pH 7.0) was estimated from the ratio of the NADPH and NADP+ rate constants corresponding to the ping-pong mechanism.     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

Cardiac-restricted overexpression of extracellular matrix metalloproteinase inducer causes myocardial remodeling and dysfunction in aging mice

The matrix metalloproteinases (MMPs) play a pivotal role in adverse left ventricular (LV) myocardial remodeling. The transmembrane protein extracellular MMP inducer (EMMPRIN) causes increased MMP expression in vitro, and elevated levels occur in patients with LV failure. However, the direct consequences of a prolonged increase in the myocardial expression of EMMPRIN in vivo remained unexplored. Cardiac-restricted EMMPRIN expression (EMMPRINexp) was constructed in mice using the full-length human EMMPRIN gene ligated to the myosin heavy chain promoter, which yielded approximately a twofold increase in EMMPRIN compared with that of the age/strain-matched wild-type (WT) mice; EMMPRINexp (n=27) and WT (n=33) mice were examined at 3.2+/-0.1 or at 13.3+/-0.5 mo of age (n=43 and 26, respectively). LV end-diastolic volume (EDV) was similar in young EMMPRINexp and WT mice (54+/-2 vs. 57+/-3 microl), but LV ejection fraction (EF) was reduced (51+/-1 vs. 57+/-1%; P<0.05). In old EMMPRINexp mice, LV EDV was increased compared with WT mice values (76+/-3 vs. 58+/-3 microl; P<0.05) and LV EF was significantly reduced (45+/-1 vs. 57+/-2%; P<0.05). In EMMPRINexp old mice, myocardial MMP-2 and membrane type-1 MMP levels were increased by >50% from WT values (P<0.05) and were accompanied by a twofold higher collagen content (P<0.05). Persistent myocardial EMMPRINexp in aging mice caused increased levels of both soluble and membrane type MMPs, fibrosis, and was associated with adverse LV remodeling. These findings suggest that EMMPRIN is an upstream signaling pathway that can play a mechanistic role in adverse remodeling within the myocardium.     

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.)

The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P=0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24h to a dose of benzo[a]pyrene (30 microg per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo.     

Seasonal variation in the frequency of abnormal anaphases and mitotic index values in wild populations of herb-Paris (Paris quadrifolia L., Trilliaceae): implications for genetic monitoring

The main aim of our study was to investigate seasonal variation in the frequency of abnormal anaphases and mitotic index values in wild populations of herb-Paris (Paris quadrifolia L., Trilliaceae). Plant material was collected in the year 2000 in Norway and in the year 2001 in Lithuania. There was statistically significant variation in the mitotic index values (chi(2)=1087.9, d.f.=16, P<0.0001) with the highest values during the active growth period in May and the lowest values at the end of vegetation period in September. Seasonal variation in the frequency of abnormal anaphases was statistically significant as well (chi(2)=28.23, d.f.=16, P=0.0297). The most frequent type of anaphase abnormality was vagrant chromosomes (64.2%) followed by bridges (28.6%), fragments (3.6%), sticky chromosomes (2.4%) and multipolar anaphases (1.2%). During the fieldwork, quite deep late frosts occurred. Mitotic index was lower in the plants collected immediately after the frosts or 1 week later than in the plants sampled before the frosts (52+/-13 and 123+/-15, respectively, P=0.0014). On the contrary, frequency of abnormal anaphases was statistically significantly elevated (P=0.0082) in plants after the frosts (6.35+/-1.54%) when compared to plants before the frosts (2.49+/-0.56%). Our results clearly indicated significant variation in the mitotic index values and frequency of abnormal anaphases in the wild populations of herb-Paris during the growth season. This variation may be related to the physiological conditions of the analysed plants as well as to certain ecological factors.     

Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds

1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an "outer-sphere" type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.