A new, sensitive method for enzyme kinetic studies of scarce glucosides

The maize β-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates.We developed a glucose assay using a system comprising three enzymes β-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-β-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.     

Blood lipid profile and myocardial superoxide dismutase in swim-trained young and middle-aged rats: comparison between left and right ventricular adaptations to oxidative stress

Region-wise interactive effects of age, swim intensity, and duration on exercise performance in the myocardium and serum lipid profile in young (4 months) and middle-aged (12 months) rats were examined. Animals were allocated to the sedentary control (SE-C) or one of the nine trainee groups. Swim training was for 6 days/week and for 4 weeks at 3 durations (20, 40, and 60 min/day) and intensities (2%, low; 3%, medium; 5%, high). Swim velocity and external work showed an age-related decline with low-intensity of 20 min/day in the middle aged. Reduction in serum cholesterol, low-density lipoproteins (LDLs), and triglycerides were accompanied by elevated levels in high-density lipoprotein in the low-to-moderately trained ones for 20 and 40 min/day. Training at 2%, intensity for 20 min/day was sufficient to alter the blood lipid profile and improve swim performance, and endurance in terms of blood lactate. A concomitant increase in Mn-superoxide dismutase (Mn-SOD) activity and reduced malondialdehyde in the left ventricle (LV) and right ventricle (RV) were evident. Lipofuscin was higher in the LV compared to RV. Our results reflect the minimization of free radical generation through appropriate exercise protocols. Our findings on improved blood lipid profile could be related to lower free radicals, which would otherwise oxidize LDLs. Further, swim training when initiated in the young and middle age for as low as 20 min/day at 2% intensity improves the Mn-SOD in the LV and RV. However, the adaptive response of the LV was weaker when compared to the RV, more so in the middle aged.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

Evidence for distinct functions for human DNA repair factors hHR23A and hHR23B

Rad23 proteins bind ubiquitinated substrates and the proteasome, consistent with an important role in protein degradation. Although human Rad23 proteins (hHR23A and hHR23B) have redundant roles in DNA repair, we determined they formed distinct interactions with proteasomes and multiubiquitinated proteins, but similar binding to Ataxin-3. Threonine-79 contributed to the weak proteasome-binding property of hHR23A, and its conversion to proline (T79P), which is the residue present in hHR23B, increased proteasome interaction. We also determined that hHR23A and hHR23B could be co-purified with unique proteolytic and stress-responsive factors from human breast cancer tissues, indicating that they have unique functions in vivo.     

Antibacterial Co(II), Ni(II), Cu(II) and Zn(II) complexes of Schiff bases derived from fluorobenzaldehyde and triazoles

Antibacterial Schiff bases derived from 1,2,4-triazoles as well as their metal complexes incorporating cobalt(II), nickel(II), copper(II) and zinc(II) have been synthesized and characterized. Physico-chemical studies suggest that an octahedral geometry for the cobalt(II), nickel(II) and zinc(II)and square-planer geometry for the copper(II) complexes. These complexes have been screened for antibacterial activity against three Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and two Gram-negative (Salmonella typhi and Pseudomonas aeruginosa) bacterial strains, and results compared with the activity of the free ligands. The metal complexes were found to be more potent against one or more bacterial strains than the free ligands.     

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.