Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis.

The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages.     

Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ-1 sites (Ser¹⁹⁹/Ser²⁰²) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The K _m values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase-2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.     

Biotic elicitors enhance diosgenin production in Helicteres isora L. suspension cultures via up-regulation of CAS and HMGR genes

In an attempt to find an alternative and potent source of diosgenin, a steroidal saponin in great demand for its pharmaceutical importance, Helicteres isora suspension cultures were explored for diosgenin extraction. The effect of biotic elicitors on the biosynthesis of diosgenin, in suspension cultures of H. isora was studied. Bacterial as well as fungal elicitors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger were applied at varying concentrations to investigate their effects on diosgenin content. The HPLC based quantification of the treated samples proved that amongst the biotic elicitors, E. coli (1.5%) proved best with a 9.1-fold increase in diosgenin content over respective control cultures. Further, the scaling-up of the suspension culture to shake-flask and ultimately to bioreactor level were carried out for production of diosgenin. During all the scaling-up stages, diosgenin yield obtained was in the range between 7.91 and 8.64 mg l−1, where diosgenin content was increased with volume of the medium. The quantitative real-time PCR (qRT-PCR) analysis showed biotic elicitors induced the expression levels of regulatory genes in diosgenin biosynthetic pathway, the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cycloartenol synthase (CAS), which can be positively correlated with elicited diosgenin contents in those cultures. The study holds significance as H. isora represents a cleaner and easy source of diosgenin where unlike other traditional sources, it is not admixed with other steroidal saponins, and the scaled-up levels of diosgenin achieved herein have the potential to be explored commercially.     

Lessons learned from the syndrome of oculopalatal tremor

Journal of Computational Neuroscience - The syndrome of oculopalatal tremor (OPT) featuring the olivo-cerebellar hypersychrony leads to disabling pendular nystagmus and palatal myoclonus. This rare...     

Photodegradation and Stabilization of Betamethasone-17 Valerate in Aqueous/Organic Solvents and Topical Formulations

The effects of solvent [acetonitrile, methanol, and acetonitrile/water mixture (20:80, v/v)], buffer concentration (phosphate buffer, pH 7.5), ionic strength and commonly employed adjuvants on the photodegradation of betamethasone-17 valerate in cream and gel formulations have been studied on exposure to UV light (300–400 nm). A validated high-performance liquid chromatography method has been used to determine the parent compound and its photodegraded products. The photodegradation data in the studied solvents showed greater decomposition of the drug in solvents with a lower dielectric constant. A comparatively higher rate of photodegradation was observed in the cream formulation compared to that for the gel formulation. The kinetic treatment of the photodegradation data revealed that the degradation of the drug follows first-order kinetics and the apparent first-order rate constants for the photodegradation reactions, in the media studied, range from 1.62 to 11.30 × 10⁻³ min⁻¹. The values of the rate constants decrease with increasing phosphate concentration and ionic strength which could be due to the deactivation of the excited state and radical quenching. The second-order rate constant (k′) for the phosphate ion-inhibited reactions at pH 7.5 has been found to be 5.22 × 10⁻² M⁻¹ s⁻¹. An effective photostabilization of the drug has been achieved in cream and gel formulations with titanium dioxide (33.5–42.5%), vanillin (21.6–28.7%), and butyl hydroxytoluene (18.2–21.6%).Key words: betamethasone-17 valerate, creams and gels, kinetics, photodegradation, photostabilization     

CCAAT/enhancer-binding protein beta (C/EBPβ) knockdown reduces inflammation,ER stress,and apoptosis,and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells

Molecular and Cellular Biochemistry - Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPβ)...     

Fish oil disrupts MHC class II lateral organization on the B-cell side of the immunological synapse independent of B-T cell adhesion

Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4⁺ T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MβCD). MβCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.     

Employers’ perspective of workplace breastfeeding support in Karachi,Pakistan: a cross-sectional study

Background

Breastfeeding is considered to be an important measure to achieve optimum health outcomes for children, women’s return to work has frequently been found to be a main contributor to the early discontinuation of breastfeeding. The aim of the study is to assess workplace breastfeeding support provided to working mothers in Pakistan.

Method

A workplace based cross-sectional survey was conducted from April through December 2014. Employers from a representative sample of 297 workplaces were interviewed on pre-tested and structured questionnaire. The response rate was 93.7 %. Prevalence of workplace breastfeeding facilities were assessed in the light of World Alliance for Breastfeeding Action (WABA) guidelines.

Results

Among non-physical facilities, all workplaces offered 3 months paid maternity leave, 45 % of the sites were offering task adjustment to mothers during lactation period. Only 15 % of the sites were offering breastfeeding breaks to working mothers. Physical facilities that include a breastfeeding corner, refrigerator for storing breast milk, breast milk pump and nursery for childcare were provided in less than 7 % of the sites. Multinational organizations provided better support compared to national organizations.

Conclusion

Support for continuation of breastfeeding by working women at workplaces is inadequate; hence, women discontinue breastfeeding earlier than planned. Policies need to be developed and enforced, employers and employees need to be educated and supportive environment needs to be created to encourage and facilitate breastfeeding friendly worksite environment.