Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10⁴ M⁻¹. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H₂O₂.     

Computer modeling of mitochondrial tricarboxylic acid cycle, oxidative phosphorylation, metabolite transport, and electrophysiology

A computational model of mitochondrial metabolism and electrophysiology is introduced and applied to analysis of data from isolated cardiac mitochondria and data on phosphate metabolites in striated muscle in vivo. This model is constructed based on detailed kinetics and thermodynamically balanced reaction mechanisms and a strict accounting of rapidly equilibrating biochemical species. Since building such a model requires introducing a large number of adjustable kinetic parameters, a correspondingly large amount of independent data from isolated mitochondria respiring on different substrates and subject to a variety of protocols is used to parameterize the model and ensure that it is challenged by a wide range of data corresponding to diverse conditions. The developed model is further validated by both in vitro data on isolated cardiac mitochondria and in vivo experimental measurements on human skeletal muscle. The validated model is used to predict the roles of NAD and ADP in regulating the tricarboxylic acid cycle dehydrogenase fluxes, demonstrating that NAD is the more important regulator. Further model predictions reveal that a decrease of cytosolic pH value results in decreases in mitochondrial membrane potential and a corresponding drop in the ability of the mitochondria to synthesize ATP at the hydrolysis potential required for cellular function.     

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: implications in excitotoxicity

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by beta-N-oxalyl amino-L-alanine (L-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. L-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or L-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with L-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to L-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to L-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

A novel approach to cyclin-dependent kinase 5/p25 inhibitors: A potential treatment for Alzheimer's disease

Based on the earlier results of our in-house database and compound library, a series of novel clubbed thienyl triazoles was designed which may emerge as potential cdk5/p25 inhibitors, for the treatment of Alzheimer's disease. A benign synthesis was planned so as to take an advantage of MAOS (Microwave Assisted Organic Synthesis) method. Evaluation of the SAR of this series has allowed the identification of compounds 4, 5, 7 and 8 from series I while 13, 14, 16 and 17 from series II as significant cdk5/p25 inhibitors and thus have potential as possible treatments for Alzheimer's disease.     

Inhibition of Ribonuclease A by polyphenols present in green tea

We report the effect of the natural polyphenolic compounds from green tea on the catalytic activity of Ribonuclease A (RNase A). The compounds behave as noncompetitive inhibitors of the protein with inhibition constants ranging from 80-1300 microM. The dissociation constants range from 50-150 microM for the RNase A-polyphenol complexes as determined by ultraviolet (UV) and circular dichroism (CD) studies. We have also investigated the changes in the secondary structure of RNase A on complex formation by CD and Fourier transformed infrared (FTIR) spectroscopy. The presence of the gallate moiety has been shown to be important for the inhibition of enzymatic activity. Docking studies for these compounds indicate that the preferred site of binding is the region encompassing residues 34-39 with possible hydrogen bonding with Lys 7 and Arg 10. Finally we have also looked at changes in the accessible surface area of the interacting residues on complex formation for an insight into the residues involved in the interaction.     

datPAV--an online processing, analysis and visualization tool for exploratory investigation of experimental data

SUMMARY: Data processing, analysis and visualization (datPAV) is an exploratory tool that allows experimentalist to quickly assess the general characteristics of the data. This platform-independent software is designed as a generic tool to process and visualize data matrices. This tool explores organization of the data, detect errors and support basic statistical analyses. Processed data can be reused whereby different step-by-step data processing/analysis workflows can be created to carry out detailed investigation. The visualization option provides publication-ready graphics. Applications of this tool are demonstrated at the web site for three cases of metabolomics, environmental and hydrodynamic data analysis. AVAILABILITY: datPAV is available free for academic use at http://www.sdwa.nus.edu.sg/datPAV/.