Assays of urea and arginase with a biocatalytic membrane electrode operating on conductimetric principle

A bioelectrode consisting of two parallel noble metal nets and a thin layer of gel-entrapped urease between them permits to determine urea conductimetrically with a response time 3–5 min and reproducibility 5–6% above 60 micromolar concentration of urea. Differential measurement eliminates the interference due to conductive components of the real sample. Evidence has been obtained for extended application of the method by using immobilized microbial cells or tissue slice as biocatalyst in place of the immobilized purified enzyme. It is also suitable for simple kinetical assaying arginase activity in the tissue extracts.     

Neuroprotective Effects of Lacosamide and Memantine on Hyperoxic Brain Injury in Rats

Neurochemical Research - In neonates supraphysiological oxygen therapy has been demonstrated to cause neuronal death in hippocampus, prefrontal cortex, parietal cortex, and retrosplenial cortex....     

Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

Leishmania infantum causes Visceral and cutaneous leishmaniasis in northern Morocco. It predominantly affects children under 5 years with incidence of 150 cases/year. Genetic variability and population structure have been investigated for 33 strains isolated from infected dogs and humans in Morocco. A multilocus microsatellite typing (MLMT) approach was used in which a MLMtype based on size variation in 14 independent microsatellite markers was compiled for each strain. MLMT profiles of 10 Tunisian, 10 Algerian and 21 European strains which belonged to zymodeme MON-1 and non-MON-1 according to multilocus enzyme electrophoresis (MLEE) were included for comparison. A Bayesian model-based approach and phylogenetic analysis inferred two L.infantum sub-populations; Sub-population A consists of 13 Moroccan strains grouped with all European strains of MON-1 type; and sub-population B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses sub-populations were significantly different from each other and from the Tunisian, Algerian and European non MON-1 strains which constructed one separate population. The presence of these two sub-populations co-existing in Moroccan endemics suggests multiple introduction of L. infantum from/to Morocco; (1) Introduction from/to the neighboring North African countries, (2) Introduction from/to the Europe. These scenarios are supported by the presence of sub-population B and sub-population A respectively. Gene flow was noticed between sub-populations A and B. Five strains showed mixed A/B genotypes indicating possible recombination between the two populations. MLMT has proven to be a powerful tool for eco-epidemiological and population genetic investigations of Leishmania.     

A generating model of realistic synthetic heart sounds for performance assessment of phonocardiogram processing algorithms

A new model which is capable of generating realistic synthetic phonocardiogram (PCG) signals is introduced based on three coupled ordinary differential equations. The new PCG model takes into account the respiratory frequency, the heart rate variability and the time splitting of first and second heart sounds. This time splitting occurs with each cardiac cycle and varies with inhalation and exhalation. Clinical PCG statistics and the close temporal relationship between events in ECG and PCG are used to deduce values of PCG model parameters.In comparison with published PCG models, the proposed model allows a larger number of known PCG features to be taken into consideration. Moreover it is able to generate both normal and abnormal realistic synthetic heart sounds. Results show that these synthetic PCG signals have the closest features to those of a conventional heart sound in both time and frequency domains. Additionally, a sound quality test carried out by eight cardiologists demonstrates that the proposed model outperforms the existing models.This new PCG model is promising and useful in assessing signal processing techniques which are developed to help clinical diagnosis based on PCG.     

The Effect of Boron on Some Biochemical Parameters in Experimental Diabetic Rats

In this study, we evaluated the effect of boron (B) as boric acid (BA) on body weight (b.w.); blood glucose; plasma insulin; lipase and paraoxonase (PON1) activities; and serum triglyceride, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, lipid peroxidation (MDA), and total antioxidant capacity (TAC) in streptozotocin (STZ)-induced experimental diabetes in rats. Sixty Wistar albino rats (200–250 g) were divided into six groups of ten. The groups received the following treatment: group 1, control group; group 2, 50 mg/kg (b.w.) i.p. STZ-induced diabetes; group 3, 5 mg/kg (b.w.) B; group 4, 10 mg/kg (b.w.) B; group 5, diabetes + 5 mg/kg (b.w.) B; and group 6, diabetes + 10 mg/kg (b.w.) B. The experiment lasted 4 weeks. Increased serum MDA levels with diabetes were significantly reduced and although it is not statistically significant, serum TAC levels approached to values of control group; also, insignificant increases were observed in HDL cholesterol levels in experimental diabetic rats with treatment 5 and 10 mg/kg B. Furthermore, body weight, plasma insulin, and lipase activities increased insignificantly, blood glucose and serum LDL cholesterol decreased significantly, and total cholesterol levels decreased insignificantly in the diabetes + 10 mg/kg B group. There was no difference between the groups in terms of plasma PON1 activities and serum triglyceride levels. In conclusion, B may have beneficial effects on some biochemical parameters changes in experimental diabetes, and in order to determine the full effect of this element on the metabolism, further studies are required which use various dosages and compounds of B.     

Target-specific delivery of doxorubicin to human glioblastoma cell line via ssDNA aptamer

Targeted drug delivery approaches have been implementing significant therapeutic gain for cancer treatment since last decades. Aptamers are one of the mostly used and highly selective targeting agents for cancer cells. Herein, we address a nano-sized targeted drug delivery approach adorned with A-172 glioblastoma cell-line-specific single stranded DNA (ssDNA) aptamer in which the chemotherapeutic agent Doxorubicin (DOX) had been conjugated. DNA aptamer, GMT-3, was previously selected for specific recognition of glioblastoma and represented many advantageous characteristics for drug targeting purposes. Flow cytometry analysis proved the binding efficiency of the specific aptamer to tumour cell lines. Cell-type-specific toxicity of GMT-3:DOX complex was showed by XTT assay and terminated cytotoxic effects were screened for both target cell and a control breast cancer cell line. The result of this contribution demonstrated the potential utility of GMT-3 aptamer-mediated therapeutic drug transportation in the treatment of gliomas specifically. It was concluded that aptamer-mediated drug delivery can be applied successfully for clinical use.     

Existence of a macrophyte-dominated clear water state over a very wide range of nutrient concentrations in a small shallow lake

Little Mere, a small shallow lake, has been monitored for four years, since its main source of nutrients (sewage effluent) was diverted. The lake has provided strong evidence for the persistence of a clear water state over a wide range of nutrient concentrations. It had clear water at extremely high nutrient concentrations prior to effluent diversion, associated with high densities of the large body-sized grazer, Daphnia magna, associated with low fish densities and fish predation. Following sewage effluent diversion in 1991, the nutrient concentrations significantly declined, the oxygen concentrations rose, and fish predation increased. The dominance of large body-sized grazers shifted to one of relatively smaller body-sized animals but the clear water state has been maintained. This is probably due to provision of refuges for grazers by large nymphaeid stands (also found prior to diversion). There has been a continued decrease in nutrient concentrations and expansion of the total macrophyte coverage, largely by submerged plants, following effluent diversion. The grazer community of Little Mere has also responded to this latter change with a decline in daphnids and increase in densities of weed-associated grazers. The presence of large densities of such open water grazers was the apparent main buffer mechanisms of the clear water state until 1994. The lake has, so far, maintained its clear water in the absence of such grazers. Thus, new buffer mechanisms appear to operate to stabilize the ecosystem. Little Mere appears to have shifted from previous top-down controlled clear water state to a bottom-up controlled clear water state.     

Co-immobilization of different enzyme activities to non-woven polyester surfaces

Co-immobilization was applied to combine complementary enzyme reactions. Therefore, trypsin was co-immobilized together with both, lipase and alpha-amylase, onto the surface of non-woven polyester material. The progress of the immobilization reaction was directly monitored by investigating covalent fixation of the enzymes to the polyester flees using (1)H-MAS-NMR. Co-immobilization of the different types of enzymes to the polyester support showed retained enzymatic activity. However, a competition of binding to the support was observed. Increasing amounts of one type of enzyme reduced the degree of immobilization for the other type. In order to investigate the distribution of trypsin and alpha-amylase on the polyester support, the flees was treated with a mixture of rhodamine isothiocyanate labeled with anti-trypsin antibodies and fluorescein isothiocyanate labeled with anti-alpha-amylase antibodies. Using fluorescence microscopy, the co-immobilization was analyzed by selective excitation of both chromophores at 480 and 530 nm, respectively. In addition, fluorescence spectroscopy was applied by direct labeling of trypsin and lipase prior to co-immobilization to the polyester support. A special prism of plexiglass was constructed, which fit into a 10 x 10 mm fluorescence cuvette in that way that a diagonal plane was formed within the cuvette. The non-woven support was fixed in the cuvette and fluorescence spectra were obtained to characterize the amount of different enzymes linked to the support. Using FRET it was demonstrated that a uniform distribution of the various enzyme species was achieved, where the different enzyme activities are bound on the support in close neighborhood to one another.     

Oxidative stress, metabolism of ethanol and alcohol-related diseases

Alcohol-induced oxidative stress is linked to the metabolism of ethanol. Three metabolic pathways of ethanol have been described in the human body so far. They involve the following enzymes: alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS) and catalase. Each of these pathways could produce free radicals which affect the antioxidant system. Ethanol per se, hyperlactacidemia and elevated NADH increase xanthine oxidase activity, which results in the production of superoxide. Lipid peroxidation and superoxide production correlate with the amount of cytochrome P450 2E1. MEOS aggravates the oxidative stress directly as well as indirectly by impairing the defense systems. Hydroxyethyl radicals are probably involved in the alkylation of hepatic proteins. Nitric oxide (NO) is one of the key factors contributing to the vessel wall homeostasis, an important mediator of the vascular tone and neuronal transduction, and has cytotoxic effects. Stable metabolites--nitrites and nitrates--were increased in alcoholics (34.3 +/- 2.6 vs. 22.7 +/- 1.2 micromol/l, p < 0.001). High NO concentration could be discussed for its excitotoxicity and may be linked to cytotoxicity in neurons, glia and myelin. Formation of NO has been linked to an increased preference for and tolerance to alcohol in recent studies. Increased NO biosynthesis also via inducible NO synthase (NOS, chronic stimulation) may contribute to platelet and endothelial dysfunctions. Comparison of chronically ethanol-fed rats and controls demonstrates that exposure to ethanol causes a decrease in NADPH diaphorase activity (neuronal NOS) in neurons and fibers of the cerebellar cortex and superior colliculus (stratum griseum superficiale and intermedium) in rats. These changes in the highly organized structure contribute to the motor disturbances, which are associated with alcohol abuse. Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression, and they correlate significantly with the disease severity. The low-density lipoprotein (LDL) oxidation is supposed to be one of the most important pathogenic mechanisms of atherogenesis, and antibodies against oxidized LDL (oxLDL) are some kind of epiphenomenon of this process. We studied IgG oxLDL and four APA (anticardiolipin, antiphosphatidylserine, antiphosphatidylethanolamine and antiphosphatidylcholine antibodies). The IgG oxLDL (406.4 +/- 52.5 vs. 499.9 +/- 52.5 mU/ml) was not affected in alcoholic patients, but oxLDL was higher (71.6 +/- 4.1 vs. 44.2 +/- 2.7 micromol/l, p < 0.001). The prevalence of studied APA in alcoholics with mildly affected liver function was higher than in controls, but not significantly. On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide range of IgG oxLDL titers in a healthy population. These parameters do not appear to be very promising for the evaluation of the risk of atherosclerosis. Free radicals increase the oxidative modification of LDL. This is one of the most important mechanisms, which increases cardiovascular risk in chronic alcoholic patients. Important enzymatic antioxidant systems - superoxide dismutase and glutathione peroxidase - are decreased in alcoholics. We did not find any changes of serum retinol and tocopherol concentrations in alcoholics, and blood and plasma selenium and copper levels were unchanged as well. Only the zinc concentration was decreased in plasma. It could be related to the impairment of the immune system in alcoholics. Measurement of these parameters in blood compartments does not seem to indicate a possible organ, e.g. liver deficiency.     

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide.