L-asparaginase-mediated Therapy in L-asparagine Auxotrophic Cancers: A Review.

Microbial L-asparaginase is the most effective first-line therapy used in the treatment protocols of paediatric and adult leukemia. Leukemic cells' auxotrophy for L-asparagine is exploited as a therapeutic strategy to mediate cell death through metabolic blockade of L-asparagine using L-asparaginase. Escherichia coli and Erwinia chrysanthemi serve as the major enzyme deriving sources accepted in clinical practice, and the enzyme has bestowed improvements in patient outcomes over the last 40 years. However, an array of side effects generated by the native enzymes due to glutamine co-catalysis and short serum stays augmenting frequent dosages intended a therapeutic switch towards developing bio better alternatives for the enzyme, including the formulations resulting in sustained local depletion of Lasparagine. In addition, the treatment with L-asparaginase in a few cancer types has proven to elicit drug-induced cytoprotective autophagy mechanisms and therefore warrants concern. Although the off-target glutamine hydrolysis has been viewed as contributing to the drug-induced secondary responses in cells deficient with asparagine synthetase machinery, the beneficial role of glutaminase-asparaginase in proliferative regulation of asparagine prototrophic cells has been looked forward. The current review provides an overview of the enzyme's clinical applications in leukemia and possible therapeutic implications in other solid tumours, recent advancements in drug formulations, and discusses the aspects of two-sided roles of glutaminase-asparaginases and drug-induced cytoprotective autophagy mechanisms.

Polyethylene Glycol Acts as a Mechanistic Stabilizer of L-asparaginase: A Computational Probing.

BACKGROUND: L-asparaginase (L-ASN) is an anti-cancer enzyme therapeutic drug that exerts cytotoxicity via inhibition of protein synthesis through depletion of L-asparagine in the tumor microenvironment. The therapeutic performance of the native drug is partial due to the associated instability, reduced half-life and immunogenic complications. OBJECTIVE: In this study, we attempted the modification of recombinant L-asparaginase with PEG and an integrated computational strategy to probe the PEGylation in the protein to understand the biological stability/activity imparted by PEG. METHODS: In vitro PEGylation of recombinant L-ASN was carried out and further evaluated in silico. RESULTS: PEGylation enhanced thermal and pH activities with extended serum half-life and resistance to proteases compared to the native enzyme. The molecular dynamics analysis revealed intricate interactions required in the coupling of PEG to L-asparaginase to bestow stronger binding affinity of L-asparagine moiety towards L-asparaginase. PEG-asparagine complex ensured stable conformation over both the native protein and asparagine-protein complex thus elucidating the PEG-induced stable conformation in the protein. PEG mechanistically stabilized L-asparaginase through inducing pocket modification at the receptor to adapt to the cavity. CONCLUSION: The study provides the rationale of PEGylation in imparting the stability towards Lasparaginase which would expand the potential application of L-asparaginase enzyme for the effective treatment of cancer.

Comparison of colorimetric and chemiluminescent enzyme-linked immunosorbent assay for the detection of endosulfan in food samples.

Pesticides have become part of food protection since their inception. Endosulfan, an organochlorine insecticide, has been used against insect pests such as whiteflies, aphids, red spiders and mites. Methods of immunochemical assays have been devised for the determination and analysis of pesticides and commonly used for the analysis of contaminants in food, water, soil and body fluids. Chicken IgY antibodies raised against endosulfan haptens were used for the detection of endosulfan. We have compared colorimetric (CO) and chemiluminescence (CL) enzyme-linked immunosorbent assay (ELISA) techniques for the detection of endosulfan isomers in a food matrix. CL ELISA assay was found to be more sensitive than CO assay. The mean recovery was 81.2-95.6% for α- and ß-endosulfan-spiked food samples with 2.8-4.6% relative standard deviation. The detection of the endosulfan isomers was linear in the range 100 µg/mL-5 fg/mL, with a limit of detection at 100 µg/mL and 5 fg/mL for the CL ELISA method and 100 µg/mL and 1 ng/mL for the CO ELISA method respectively. These methods can be used for the rapid and reliable detection of organochlorine pesticide endosulfan.

Degradation of isoprothiolane by a defined microbial consortium using response surface methodology.

A defined microbial consortium was developed for the degradation of isoprothiolane. Isoprothiolane-biodegradation parameters were optimized using Response Surface Methodology (RSM). Three variables chosen for the study were inoculum concentration (50-1500 microg protein ml(-1)), temperature (25-35 degrees C) and pH (4-8) each at levels -1.682, -1, 0, 1 and 1.682. Incubation time of 72 hr was kept constant. Degradation of different concentrations of isoprothiolane was studied. The optimized conditions obtained were, inoculum concentration of 50 microg protein ml(-1) at 30 degrees C and pH between 4-8. The maximum predicted percentage degradation of 100, 100, 100, 100 and 95.5 was obtained respectively for 5, 10, 20, 30 and 50 ppm of initial isoprothiolane concentrations at different pH levels 7.7, 6.8, 6.2, 4.7 and 4.6. Validation of the model indicated that experimental values were found to be in agreement with the predicted one.

Development of dipstick-based immuno-chemiluminescence techniques for the rapid detection of dichlorodiphenyltrichloroethane.

The occurrence of organochlorine pesticides in the environment has been a major concern, due to their high persistence and the possible impacts of their exposure to humans. Dichlorodiphenyltrichloroethane (DDT) is most hazardous and one of the most widely used organochlorine insecticides. DDT and its main metabolites are highly stable to physical, chemical and biological degradation and are therefore still being detected in many parts of the world. The present study describes dipstick-based immuno-chemiluminescence method for the detection of DDT with high sensitivity. Anti-DDT antibodies raised in chicken (IgY) were used as the biological sensing elements by immobilizing onto nitrocellulose membrane strips in a chemiluminescence (CL)-based dipstick technique. The photons generated during the biochemical interaction were directly proportional to the DDT concentration. A mean recovery of 81.2-95.6% was obtained for DDT-spiked fruit juice samples with 2.8-4.6% relative standard deviation (RSD). Using the proposed dipstick-based immuno-CL method, DDT was detected with linearity in the range 0.05-1 ng/mL, having a limit of detection (LOD) of 0.05 ng/mL. This method can be used for the rapid, reliable detection of DDT pesticide.