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Evaluation of Genetic Association of the INK4 Locus with Primary Open Angle Glaucoma in East Indian Populati
No full textINK4 locus at chromosome 9p21 has been reported to be associated with primary open angle glaucoma (POAG) and its subtypes along with the associated optic disc parameters across the populations of European, Japanese and African ancestries. The locus encodes three tumor suppressor genes namely CDKN2A, ARF, CDKN2B and a long non-coding RNA CDKN2B-AS1 (also known as ANRIL). Here, we report association study of 34 SNPs from INK4 locus withPOAGin a population of Indo-European ancestry from the eastern part of India (350 patients and 354 controls). With 81% power to detect genetic association we observed only nominal association of rs1011970 (uncorrected p50.048) with POAG and rs10120688
(uncorrected p50.048) in patients without a high intra-ocular pressure (IOP,21 mm of Hg) compared to controls. This study, in contrast to the previous reports, suggests lack of significant genetic association of INK4 locus with POAG in East Indian population which needs to be replicated in larger studies in diverse world populations
IL-4 contributes to failure, and colludes with IL-10 to exacerbate Leishmania donovani infection following administration of a subcutaneous leishmanial antigen vaccine
Get PDFVisceral leishmaniasis caused by the protozoan parasite Leishmania donovani complex is a potentially
fatal disease if left untreated. Few treatment options exist and are toxic, costly and ineffective against resistant
strains. Thus a safe and efficacious vaccine to combat this disease is needed. Previously, we reported that intraperitoneal
administration of leishmanial antigens (LAg) entrapped in liposomes conferred protection to BALB/c mice against
L. donovani challenge infection. However, this vaccine failed to protect mice when administered subcutaneously. We
therefore evaluated whether formulation of LAg in combination with two commonly used human-compatible
adjuvants, alum and saponin, could improve the protective efficacy of subcutaneously administered LAg, to a level
comparable to that of the intraperitoneal liposomal vaccinatio
Role And Regulation Of Hausp In Cancer
No full textCancer is a disease of uncontrolled cell growth resulting from deregulated cellular signaling. Signal transduction is subject to several modes of regulation.This study is focussed on post-translational modifications, especially
deubiquitination. The thesis work is divided into four sections, the first three emphasize on the role of HAUSP and the fourth one is on the regulation of
HAUSP.In the first part of the work, we have found a large number of putative substrates of HAUSP by MALDI-TOF/TOF MS/MS analysis. We have shown the effects of exogenously expressed HAUSP on the overall proteome. To further
investigate the interacting partners of HAUSP, the pool of interacting proteins was enriched by immunoprecipitation. Gene ontology based analysis of the proteins
identified, indicate a probable involvement of HAUSP in tumor suppression, metabolism, cytoskeletal organization and transport, gene expression, chaperone system, apoptosis, etc. A few uncharacterized/ novel proteins have also been identified. From the analysis, three putative bona fide substrates – TRRAP, Keratin and FBF-1 have been identified which were also found to colocalize with
HAUSP. In this part of the work we have shown that HAUSP plays an important role in cancer by regulating cMyc via TRRAP.In the second part, we have identified Rb as a novel substrate for HAUSP mediated deubiquitination. This has added an edge to the MDM2 – p53 mediated functions of HAUSP. We have shown the role of HAUSP in preferential
stabilization of MDM2 over Rb in cancer/ GBM cell lines. In delineating the mechanism of the phenomena, we show how HAUSP stabilizes Rb in a contextPreface dependent manner, using a p53-independent switching mechanism in normal and
cancer cells, where MDM2 is the critical regulator.
In the third part, we have identified the role of HAUSP specifically in glioma. In clinical specimens of glioma we have shown that HAUSP protein levels increase during progression from lower to higher grad(immunohistochemistry). This is very well corroborated by the in vitro data showing knockdown of HAUSP leads to huge decrease in the cellular proliferation as seen in U87MG cells while upregulation of HAUSP led to larger
sized and more number of colonies in a colony formation assay.In the fourth and last part of the work on regulation of HAUSP, first we found that in U87MG (GBM) cells HAUSP is regulated by EGF stimulation, wherein HAUSP transcript levels show a huge increase upon EGF treatment and
at the protein level we see a clear nuclear exclusion within a short period of induction. The most appropriate explanation here at this point of time would be
that in U87MG cells which harbor wild type p53, HAUSP nuclear exclusion would lead to destabilization of p53 in the nucleus, thus preventing its function as
a tumor suppressive transcription factor. Upon in silico analysis of the HAUSP promoter sequence we identified a large number of p53 binding sites giving rise to
the possible existence of a feedback regulation of HAUSP by p53. Additionally the presence of TCF4/LEF sites is also suggestive of a role of Wnt signaling in regulating HAUSP
Synthesis of Polynuclear Heteroaromatics Using Green Chemistry Protocols and their Bio-Evaluation
No full textThe work embodied in this thesis describes the facile synthesis of nitrogen containing heterocyclic compounds through facile and environment friendly protocol and evaluation of their bioactive potential. Chapter I includes a brief discussion on the development of spermicides from different natural and synthetic sources. The review is a compilation of the promising molecules and even extracts, and includes an outline of the basic physiology behind the bioactivity. Chapter II describes the facile one-pot green methodology to synthesize a series of quinoline derivatives starting from 8-OH-quinoline using di-bromo alkanes as reactant. The synthesized compounds were assessed for their sperm-immobilizing as well as microbicidal activities by various standardized assays. Mode of actions was also investigated by electron microscopic imaging and biochemical assays. Chapter III illustrates the synthesis of 7-Aryl substituted furo[3,2-h]quinoliniums from 5-chloro-8-hydroxy-7-iodo-quinoline in two steps through a tandem Sonogashira alkynylation-cyclization pathway using aryl acetylenes, followed by quaternisation reaction with alkyl halides under microwave irradiation. These compounds too have been screened and investigated for spermicidal activity.Chapter IV narrates the Friedel-Crafts reaction of differently substituted anilines with ninhydrin in presence of molecular iodine at ambient temperature, constituting a facile, cost effective, and regioselective synthesis of a series of 2-mono/2,2-bis-(amino-phenyl)-indane-1,3-dione derivatives. These compounds were screened for Microbicidal activities against quite a few number of bacterial and fungal strains
Role of Apoptosis, Autophagy and Mitochondrial Dysfunction in Anti Leukemic Activities of Andrographolide and indole derivatives
No full textCancer, the medical term of which is malignant neoplasm, is a class of disease in which a group of cells display uncontrolled growth (division beyond normal limits), invasion (intrusion on and destruction of adjacent tissues), and metastasis (spread to other locations in the body via lymph or blood). These three properties differentiate them from benign tumors which do not intrude into other regions (Merlo et al., 2006). According to the World Health Organization, cancer is the leading cause of death in developed countries and is second to cardiovascular diseases in developing countries. The total number of registered cases of cancer in the year 2010 was 979,786 of which 555,000 were cases of reported deaths (Takiar et al., 2010). International Agency for Research on Cancer estimates a whopping number of 16 million new cases of cancer by the year 2020. WHO also projects that cancer will soon replace ischemic heart disease as the overall leading cause of death worldwide (IARC press release, 2010-2020)
Novel Crosstalks in Stat3 Regulation Involving Crucial Signaling Pathways: Implications in Oncogenesis
No full textCancer, a group of diseases caused by unregulated cell growth, affects multiple body organs. Specific cancer originates from a definite organ but is not restricted to that particular region as the cells rapidly proliferate and metastasize to other distant organs by invasion or migration through blood vessels, lymphatic channels and bones. Deregulation of crucial signaling pathways in cells is thought to be the major reason for cancer initiation and progression. Among the various types of cancer, we studied the regulation of signaling of Stat3 in prostate cancer (PCa, sixth global cause of cancer related death in men) and glioblastoma multiforme (GBM) (most aggressive and malignant form of brain tumor in human with very short patient survival). Amongst the many causal reasons of PCa and GBM, alterations and perturbations in the molecular signaling pathways play a major role. Several signaling pathways have been implicated in their progression including phosphoinositide-3-kinase (PI3K), Wnt-β-catenin signaling, Ras-MAPK and tyrosine kinase signaling pathways viz. EGFR and VEGFR. The work described in this thesis is divided into three main sections, the first and second parts deal with the cross-regulation of Stat3 with other signaling pathways and the third explores the possible effect of this regulation of Stat3 on tumorigenesis. In the first part of the work, the cross-regulation between Stat3 activation and canonical Wnt/β-catenin signaling is investigated. We found that EGFR mediated activation of Stat3 resulted in over expression and nuclear accumulation of β-catenin. On the other hand, LiCl mediated stabilization of β-catenin resulted in upregulation of Stat3 in PCa. This feedback regulation between two important signaling pathways results in enhanced tumorigenesis. In the second part, regulation of Stat3 Ser-727 phosphorylation by CK2 was probed. In our study of human glioma patient samples and a rat orthotopic tumor model, Stat3 Ser-727 phosphorylation was found to occur less frequently when compared to casein kinase 2 (CK2), a well known kinase involved in cancer progression. In glioma, increased Stat3 Ser-727 phosphorylation was observed upon CK2 inhibition. Overexpression of CK2 (α, α’ or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. As CK2 hyperactivates Akt by phosphorylation a Ser-129, the role of phospho-Akt Ser-129 in Stat3 Ser-727 phosphorylation was further investigated and determined that Akt mediated regulation of Stat3 Ser-727 was independent of CK2 involvement. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased proliferation and invasion which are characteristics of a cancer cell. Thus, in gliomas, CK2 may negatively regulate Stat3 Ser-727 phosphorylation thereby enhancing tumorigenicity. In the last part, we establish the effect of reduced Stat3 Ser-727 phosphorylation in vitro and in in vivo tumor models. In the in vitro system, stable cell lines constitutively expressing Stat3 S727A mutant (Stat3 mut) showed increased cell survival, increased rate of proliferation and invasion, when compared to either Stat3 WT or empty vector containing cells. Rat tumor models injected with the Stat3 mut cells formed more aggressive tumors when compared to the Stat3 WT or empty vector cells. Thus, the inhibitory effect of Ser-727 phosphorylation on Stat3 activation is reduced in Stat3 mut cells thereby leading to more aggressive tumor formation. Targeting Stat3 activity has been the method of choice for therapies against cancer and our study provides novel axes for exploration in this direction
Regulatory studies of proteases from filamentous fungus termitomyces clypeatus
No full textOwing to immense physiological, biotechnological, biomedical as well as commercial importance, currently fungal proteolytic enzymes draw the attention of researches in many ways. While studying production of some glycosidases from the fungus Termitomyces clypeatus MTCC-5091 mainly Sucrase, proteolytic activities were detected in the culture medium. These proteases were not studied earlier. Therefore the thesis work was undertaken with an aim to study the regulation of the protease enzyme in the fungus. The fungus produced two proteases, acidic metalloprotease (AcP) and alkaline serine protease (AkP). Secretion of AcP was constitutive while AkP production was inducible and substantially dependent on external protein supplements into media. Both these proteases were monomeric and non-glycoprotein in nature. Though biochemical, kinetic properties and application potencies were appreciably different for AcP and AkP. Both the enzymes were purified to homogeneity and characterized biochemically. AcP showed high milk-clotting potency that preferentially hydrolyzed the peptide bond in Phe105-Met106 of k-casein, analyzed by Urea-PAGE and LC-ESI-MS. Purified AcP (29 kDa) had pI value 4.6 and optimally active at pH 5 and 45oC. One-and two dimensional zymographies revealed a single polypeptide band with proteolytic signal. The enzyme can be developed as a substitute for chymosin. The thesis works report the production, optimization, purification and applications of AkP from T.clypeatus focusing on bioremediation and biomedical area. AkP titre was successfully optimized through the Plackett-Burman and RSM using the Box-Behnken design. As an ecofriendly alternative, AkP showed significant promise for bioremediation and industrial applications through time-saving bioprocesses in reduction of BOD and COD values of direct CETP inlet of tannery wastes, goat hide dehairing and bird feather detachment. The sequence of first 15 N-terminal amino acids of AkP (33 kDa) showed high homology with other serine proteases. AkP had pI value 8.9 with optimal activity achieved at pH 10 and 45oC. Studies were also performed for investigating in vitro antiproliferative potency of AkP on human HepG2 cancer cells. AkP treatment resulted in G2/M arrest and apoptosis in HepG2 cells. Hence, the edible mushroom Termitomyces clypeatus is a new source of two important proteases that can be explored in future for wideranging application potential
Immunomodulatory effects of antileishmanial drugs
No full textThe commonly used antileishmanial drugs are sodium antimony gluconate (SAG), amphotericin B,
miltefosineandparomomycin. Thereareanumberof reports that antileishmanial drugsshowimmunomodulatory
properties. Here,we attempt to understand howthe innatearmof theimmunesystemis modulated in response to
these antileishmanial drugs.
BALB/c peritoneal macrophages were treated with miltefosine, SAG, amphotericin B or paromomycin.
Themembrane fluidity of macrophages following drug treatmentwas studied in terms of fluorescence anisotropy.
The T cell-stimulating ability, production of cytokines and nitrogen and oxygen metabolite production in drugtreated
macrophages were also studied. The study was also carried out using peritoneal macrophages from drug-treated BALB/c mice.
The antileishmanial drugs altered macrophage membrane fluidity, except amphotericin B. The drugtreated
macrophages showed enhanced T cell-stimulating ability and generation of reactive oxygen species, nitrite, interleukin-12 and tumour necrosis factor-a Antileishmanial drugs can stimulate the innate arm of the immune system, which may have a significant
bearing on the cellular arm of the immune system
The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: Photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction
No full textThe interaction of the natural benzophenanthridine alkaloid chelerythrine with DNA was studied by spectroscopy, viscometry and calorimetry techniques. The absorbance and fluorescence properties of the alkaloid were remarkably modified upon binding to DNA and the interaction was found to be cooperative. The mode of binding was principally by intercalation as revealed from viscosity studies and supported from fluorescence quenching, and polarization results. The binding remarkably stabilized the DNA
structure against thermal strand separation. The binding induced conformational changes in the B-form structure of the DNA and the bound alkaloid molecule acquired induced circular dichroism. The binding affinity values obtained from spectroscopy, fluorescence polarization (and anisotropy) and calorimetry were in agreement with each other. The binding was exothermic, characterized by negative enthalpy and positive entropy change and exhibited enthalpy–entropy compensation phenomenon. The heat
capacity changes of the binding revealed hydrophobic contribution to the binding. Molecular aspects of the interaction characterized by the involvement of multiple weak noncovalent forces are presented
Efficient Synthesis of Imidazole-Fused Benzodiazepines Using Palladium- Catalyzed Intramolecular C–N Bond Formation Reaction
No full textAn efficient three-step synthetic route to imidazolefused
benzodiazepines from imidazole-2-carbaldehyde is described.
Application of intramolecular Buchwald–Hartwig cycloamination
reaction in the final step is shown to be a convenient
method for the synthesis of fused seven-membered diazacycles. The
reactions proceeded smoothly with both aliphatic and aromatic
amines