Purification, and properties of a bovine uricase

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.     

Ameliorating Effect of Fish Oil on Acrylamide Induced Oxidative Stress and Neuronal Apoptosis in Cerebral Cortex

Acrylamide (ACR) is a known industrial toxic chemical that produce neurotoxicity characterized by progressive neuronal degeneration. This study was designed to investigate the protective effect of fish oil on ACR-induced neuronal damage in Wistar rats. ACR enhances the production of reactive oxygen species and potentially affects brain. ACR administered rats showed increased levels of lipid peroxidative product, protein carbonyl content, hydroxyl radical and hydroperoxide which were significantly modulated by the supplementation of fish oil. The activities of enzymic antioxidants and levels of reduced glutathione were markedly lowered in ACR-induced rats; fish oil treatment augmented these antioxidant levels in cortex. Free radicals generated during ACR administration reduced the activities of membrane adenosine triphosphatases and acetylcholine esterase. Fish oil enhanced the activities of these enzymes near normal level. Histological observation represented the protective role of fish oil in ACR-induced neuronal damage. Fish oil reduced the ACR-induced apoptosis through the modulation in expressions of B-cell lymphoma 2 (Bcl2)-associated X protein and Bcl2-associated death promoter. Further, fish oil increases the expression of heat shock protein 27 (Hsp27) in ACR-induced rats. This study provides evidence for the neuroprotective effect of fish oil on ACR-induced neurotoxicity by reducing oxidative stress and apoptosis with modulation in the expression of Hsp27.     

A kinetic zipper model with intrachain interactions applied to nucleic acid hairpin folding kinetics

Single-stranded DNA and RNA hairpin structures with 4-10 nucleotides (nt) in the loop and 5-8 basepairs (bp) in the stem fold on 10-100 μs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ~2-nt) is 10-100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔG(nWC)) and the rate at which a basepair is formed adjacent to an existing basepair (k(bp)(+)). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4-20-nt-long poly(dT) loops, with ΔG(nWC) ≈ -2.4 kcal/mol and k(bp)(+) ≥ (1 ns)(-1), in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays

An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.     

Effect of NaCl concentrations in irrigation water on growth and polyamine metabolism in two citrus rootstocks with different levels of salinity tolerance

Six-months-old, uniform sized seedlings of two citrus rootstocks; Cleopatra mandarin (Citrus reshni Hort. ex Tan) and Troyer citrange (Poncirus trifoliata × Citrus sinensis) were irrigated with half-strength Hoagland nutrient solution containing 0, 40 or 80 mM NaCl for 12 weeks. Shoot height, leaf number and fresh weights of the seedlings, and relative chlorophyll contents, chlorophyll fluorescence yields (Fv/Fm), net photosynthetic and respiration rates in the leaves decreased with the increase in salinity level in the irrigation water. The decrease was greater in Troyer citrange as compared to Cleopatra mandarin. The concentrations of sugars i.e. fructose, glucose and sucrose in the leaves of Cleopatra mandarin and both leaves and roots of Troyer citrange decreased with the increase in salinity level. However, the concentrations in the roots of Cleopatra mandarin increased with the increase in salinity level. Free proline content in the leaves of Troyer citrange and root tissue of Cleopatra mandarin also increased with the increased salinity level. Among the polyamines, spermine titer increased in the leaves of both rootstocks as a response to salinity treatments. Na⁺ concentrations were higher in leaf and root tissue of Cleopatra mandarin, while that of Cl⁻ were higher in Troyer citrange.     

Analysis of Cell Wall Constituents of Biocide-Resistant Isolates from Dental-Unit Water Line Biofilms

In past years, the significance of microbial resistance to biocides has increased. Twenty biocide-resistant bacterial strains were isolated from dental-unit water line biofilm. All strains resisted high biocide concentrations (up to 100 mug ml(-)1): sodium dodecyl sulphate, hydrogen peroxide, sodium hypochlorite, phenol, Tween 20, ethylenediaminetetraacetic acid, chlorohexidine gluconate, and povidine iodine. Among bacteria, biocide sensitivity is based on permeability of biocides through the cell wall. Gram-positive bacteria are more permeable and susceptible to biocides, whereas Gram-negative bacteria have a more complex cell wall and are the least sensitive bacteria. The present study was designed to study the effect of biocides on the cell wall of biocide-resistant bacteria. Peptidoglycan (PG), diaminopimelic acid (DAP), and teichoic acid contents of the cell wall were determined in L-broth and L-broth supplemented with biocides at different temperatures (37 degrees C and 45 degrees C) and pH levels (7 and 9). In general and Gram staining-specific comparison, a significant increase (p < 0.05) in the DAP content of biocide-resistant bacteria was observed at pH 7 and at both temperatures. In tubing-specific comparison, a significant increase in the amount of teichoic acid in air water tubing (37 degrees C at pH 9) and DAP in patient tubing (pH 7 at both temperatures) was observed. In main water pipe, a significant decrease (p > 0.05) in PG content was noticed at 45 degrees C and pH 9. Overall, a significant increase in DAP content may be an important constituent in the manifestation of isolate resistance against various biocides.     

Cefotaxime Resistant Escherichia coli Collected from a Healthy Volunteer; Characterisation and the Effect of Plasmid Loss

In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla _CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla _CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.