Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.     

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Uridine enhances expression of the fragile X chromosome in human lymphocytes

Summary We have studied the effect of uridine on the expression of fragile X (fra[X]) in lymphocyte cultures established in the folate and thymidine deficient medium TC199. The results indicate that uridine enhances the expression of fra(X) and gives a higher mitotic rate. The excess of uridine during DNA synthesis might further promote the previously suggested cycle of misincorporation and removal of deoxyuridine monophosphate when the pool of deoxythymidine triphosphate is continuously depleted.     

Polymorphic restriction sites of type II collagen gene: their location and frequencies in the Finnish population

Restriction fragment length polymorphism (RFLP) of the cartilage-specific type II collagen gene has been studied in the Finnish population. Two high-frequency alleles, also reported in other populations, were detected. The HindIII allele had a frequency of 0.33, and that detected with PvuII a frequency of 0.46. Both of these frequencies resembled the ones reported for other populations. Also one BamHI allele, not earlier reported, was found at a low frequency. Two other previously reported polymorphisms for BamHI and EcoRI were not detected in the Finnish population. The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. A linkage disequilibrium was found between PvuII and HindIII markers. The alpha 1(II) collagen gene seems to be more conserved in populations of various origins than the alpha 2(I) collagen gene. These polymorphic collagen markers would be useful in linkage studies of various inherited cartilage disorders.     

Change in survival of cells in the first 2 generations after irradiation and exposure to inhibitors of repair synthesis

In experiments on asynchronous population of HeLa S3 cells a study was made of the possibility of assessing DNA lesions which remained unrepaired for a long period of time following gamma-irradiation: in generation "O" directly affected by radiation and in generation "I" following the irradiated one. The presence of DNA damages was estimated by the reduction in survival of exposed cells incubated with inhibitors of repair and replicative syntheses of DNA, namely, with arabinoside cytosine and hydroxyurea. A considerable enhancement of the radiation effect was noted with the inhibitors added 0-6 h after irradiation (generation "O"), and a marked increase in the cell death was registered with the preparations injected 24-30 h after exposure (generation "I"). It is assumed that minor residual lesions persist in the generation of cells, following the one directly affected by gamma-radiation, which have completed the first postirradiation mitosis.     

Long-term DNA damage and mammalian cell survival

On the basis of our own data and those reported in the literature we have made an attempt to follow the fate of the DNA lesions which remain unrepaired during a long period of time, and their possible role in the fate of irradiated cells. The presence of long-lived ("residual") damages is determined by the changes in survival of exposed cells treated, at different times after irradiation, with a mixture of arabinoside cytosine and hydroxyurea. It is shown that "residual" damages can probably exist in the exposed generation and be retained in that following the irradiated one, i.e. after the first mitosis. The nearest descendants of exposed cells (the 3d-5th generations) exhibit a 50% decrease in the rate of DNA synthesis and fall of their proliferative activity, as well as a decrease in the rate of reproduction of their remote descendants. The comparison of the results obtained with those reported by other authors enable us to assume that "residual" DNA lesions play an important role in the fate of exposed cells, that is, in reproductive death, radiation mutagenesis, and malignant transformations.     

Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method

Summary Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.     

Relationship between serum lipids, lipoproteins and pseudocholinesterase during organophosphate poisoning in rabbits

The activities of serum pseudocholinesterase, lecithin: cholesterol acyltransferase (LCAT) and gamma-glutamyltransferase in rabbits were investigated before and after dichlorvos administration in vivo. The effects of this organophosphate on some serum lipids and lipoprotein fractions were also determined. LCAT activity remained almost unaffected after organophosphate administration. However, serum gamma-glutamyltransferase and pseudocholinesterase activities markedly decreased. Dichlorvos markedly lowered both serum low density lipoprotein (LDL) and cholesterol contents, whereas high density lipoprotein (HDL) concentration increased and very low density lipoprotein (VLDL) remained unaffected. Triglycerides as well as esterified fatty acids increased significantly but the statistical changes in free fatty acid concentrations were not significant, because individual variations in fatty acid concentrations were high.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.