DSpace Angular :: Browsing Biochemistry - Theses by Subject "Research Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistry"

Item

Analyasis of the functional role of microRN as in regulating macrophage mediated inflammatory responses

(University of Hyderabad, 2016-05-08) Malathi, Talari ; Kishore Parsa

Unrestrained inflammation frequently observed in chronic inflammatory diseases such as insulin resistance, Type 2 Diabetes (T2D) and several types of cancers is prominently due to imbalances in different activation states of macrophages. Delineation of the regulatory mechanisms underlying macrophage polarization may help us to better understand the pathophysiological basis of inflammation linked diseases. MicroRNAs are posttranscriptional regulatory molecules that drive distinct biological processes such as proliferation, cell survival, differentiation and inflammation. However, the functional role of microRNAs in inflammation induced insulin resistance is poorly studied. Thus, there is a necessity to study the involvement of microRNAs in inflammation induced insulin resistance (IR), as IR is the leading cause of diabetes. With this objective we sought to understand the regulatory role of microRNAs in macrophage polarization and insulin resistance. Our microRNA microarray data revealed several microRNAs that were differentially regulated in polarized macrophages. Among them, we investigated the functional roles of two microRNAs: miR-712 and miR-16 whose expression levels were down-regulated in macrophages exposed to pro-inflammatory stimuli such as LPS+IFNγ and palmitate. Additionally, we have observed that miR-16 expression is also down-regulated in palmitate exposed myoblasts, ER stress inducer (tunicamycin and thapsigargin) treated myoblasts and insulin responsive tissues of high sucrose diet (HSD) induced insulin resistant rats. On the other hand unlike in macrophages, miR-712 expression levels were observed to be increased in myoblasts upon exposure to stress inducers such as palmitate, iv tunicamycin and thapsigargin pointing to potential cell specific effects. Investigating the direct role of miR-712 in skeletal muscle may help us in better understanding of insulin mediated effects in physiology and disease. We next noted that ectopic expression of miR-712 and miR-16 in macrophages reduced the production of pro-inflammatory cytokines such as TNF-α, IL-6 and IFN-β which in turn led to improved insulin sensitivity in insulin resistant (IR) skeletal myoblasts suggesting reduced paracrine inhibitory effects of LPS+IFNγ polarized macrophages on skeletal myoblasts insulin sensitivity. In addition we observed that forced expression of miR-16 directly in myoblasts augmented insulin stimulated glucose uptake via upregulation of two key players: GLUT4 and MEF2A that are involved in insulin stimulated glucose uptake. Mechanistic analysis revealed LRRK2 (a serine / threonine protein kinase associated with inflammatory diseases such as Crohn’s and Parkinson’s disease) as the target of miR-712. Further over-expression of miR-712 resulted in reduced phosphorylation of p38 and ERK1/2, key players involved in inflammatory gene expression suggesting that miR-712 is positioned to control macrophage mediated pro-inflammatory responses. Collectively, our data demonstrates the pivotal roles of microRNAs miR-712 and miR-16 in alleviating inflammation induced insulin resistance for the first time. Exploring the pathophysiological roles of these microRNAs may further help us in understanding the progression and treatment of T2D. MicroRNA-712 and miR-16 dampen macrophage mediated pro-inflammatory responses and improve insulin mediated glucose uptake in myoblasts. (A) miR712 expression is down-regulated in LPS +IFNγ polarized macrophages. Ectopic expression of miR-712 attenuated macrophage pro-inflammatory responses and their paracrine inhibitory effects on myoblast insulin sensitivity. Mechanistic analysis showed that miR-712 directly targets LRRK2 resulting in decreased phosphorylation of p38 and ERK1/2 in macrophages. (B) miR-16 expression is down-regulated in LPS+IFNγ and palmitate stimulated macrophages and in palmitate, tunicamycin and thapsigargin treated myoblasts. Importantly, forced expression of miR-16 into macrophages improved myoblast insulin sensitivity by enhancing GLUT4 and MEF2A expression levels.

Item

Baliospermum montanum hydroxynitrile iyase catalyzed stereoselective synthesis of chiral cyanohydrins

(University of Hyderabad, 2019-04-17) Nisha Jangir ; Santosh Kumar Padhi

Optically pure cyanohydrins are potential chiral molecules used in the synthesis of various pharmaceuticals, agrochemicals and bioactive compounds. They can be synthesized by different chemical and biocatalytic methods. Enzyme mediated kinetic resolution which includes asymmetric acylation and deacylation of racemic cyanohydrins by lipases and esterases, is one of the common biocatalytic methods to obtain chiral cyanohydrins. However, kinetic resolution has the disadvantage of giving maximum 50% yield of each enantiomer. The second biocatalytic method for synthesis of chiral cyanohydrins is enantioselective C-C bond formation which is usually carried out by hydroxynitrile lyases (HNLs). Among the several HNLs known so far, the latest member of HNL from the α/ hydrolase fold is Baliospermum montanum hydroxynitrile lyase (BmHNL). It is an (S)-selective HNL with high substrate preference for bulky aromatic aldehydes. Despite of its unique substrate selectivity, BmHNL biocatalysis is limited with poor enantioselectivity. The aim of the present study is to investigate the BmHNL catalyzed stereoselective synthesis of chiral cyanohydrins to improve the enantioselectivity and stability of the enzyme in biocatalysis. To achieve the objectives, BmHNL was subcloned. Its protein expression, purification was performed and it was characterized by SDS-PAGE and enzymatic assay. Racemic cyanohydrins to be used as internal analytical standards were synthesized. Eighteen racemic cyanohydrins were synthesized using literature based and modified protocols using different cyanide donors. Among them, six racemic cyanohydrins were synthesized using KCN as cyanide donor and another six were synthesized using trimethylsilylcyanide as cyanide source. Rest six cyanohydrins were prepared by a modified protocol using v acetone cyanohydrin as source of cyanide. All the cyanohydrins were characterized by 1H and 13C NMR. Chiral resolution of racemic cyanohydrins was carried out using high performance liquid chromatography (HPLC) with Chiralpak IB and IE chiral columns. Separation factor (α) and resolution factor (Rs) of all the HPLC chromatograms of the racemic cyanohydrin were calculated. HPLC chromatograms of sixteen racemic cyanohydrins have showed α >1 and Rs > 1.5, indicting good resolution of the enantiomers. BmHNL catalyzed synthesis of (S)-cyanohydrins was carried out for the first time in a biphasic system. Toward this, standardization of reaction parameters/conditions such as different organic solvents and their percentage, substrate concentration, pH of buffer and temperature was carried out using crude enzyme. For each of the above optimization step, benzaldehyde conversion to (S)-mandelonitrile was used as the standard reaction. Using the optimized conditions, seventeen different aldehydes were converted into their corresponding (S)-cyanohydrins. In all, eight (S)-cyanohydrins reported first time here whereas fourteen were not tested for BmHNL earlier. To improve biocatalytic properties of the enzyme, BmHNL was first time immobilized using cross-linking method. Cross-linked enzyme aggregates of BmHNL i.e. CLEABmHNL was prepared and characterized by SDS-PAGE and scanning electron microscopy. The enzymatic activity recovery of CLEA-BmHNL was found to be 41.6%. The reactions conditions for CLEA-BmHNL catalyzed synthesis of (S)-cyanohydrins were optimized using benzaldehyde as a standard substrate. CLEA-BmHNL produced (S)- mandelonitrile in very high % ee i.e. 99.8. Using the optimized conditions, eleven different (S)-cyanohydrins were synthesized with good % ee. Among them, eight cyanohydrins have not been synthesized by any CLEA-BmHNL, and nine were synthesized by BmHNL for vi the first time. The reusability of CLEA-BmHNL could be reused for five cycles without loss of % ee of (S)-mandelonitrile. Effect of different biocatalytic conditions on the stability and activity of BmHNL was studied. At its optimum pH 5.5, temperature 20 oC, and buffer concentration (100 mM citrate phosphate pH 5.5) it showed half-life of 554 to 690 h, which is the maximum halflife among all α/β hydrolase fold HNLs. Addition of sucrose to BmHNL’s biocatalysis has increased its half-life by fivefold, while addition of sorbitol or glycerol increased ~ 9 fold specific activity. Among the polyols, glycerol addition to BmHNL’s biocatalysis has showed >99% ee of (S)-mandelonitrile in its synthesis. This study was extended to the synthesis of (S)- 3,5-dimethoxy mandelonitrile and (S)-3-phenoxymandelonitrile, a precursor of pyrethroid, an insecticide. Effect of organic solvents and different temperatures on secondary structure of BmHNL was studied with circular dichroism and it was observed that the secondary structure of protein was least affected by both. Keywords: Cyanohydrins, Enantoselective C-C bond formation, Baliospermum montanum hydroxynitrile lyase, Biphasic system, Immobilization, Biocatalysis, Half-life.

Item

Characterization of Mge 1, a nucleotide exchange factor of mitochondrial Hsp70, under oxidative and thermal and thermal stress

(University of Hyderabad, 2015-05-23) Adinarayana, Marada ; Naresh Babu V. Sepuri