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Exploration of Anti-cancer and Anti-inflammatory Role of Sphingolipid(s) from Attenuated Leishmania donovani
No full textSepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of Gram negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of
inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmanial donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of total lipid (LTL) isolated from Leishmanial donovani(MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional factor with suppression of
TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s) of lung injury caused by sepsis, viz. edema, vascular permeability, and patho-physiology, and the status of different cytokinechemokine(
s) and adhesion molecule(s). Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s) levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and
suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against Gram negative bacterial sepsis with pulmonary impairment
Molecular Crowding Affects the Conformational Fluctuations, Peroxidase Activity, and Folding Landscape of Yeast Cytochrome c
No full textTo understand how a protein folds and behaves inside living cells, the effects of synthetic crowding media on protein folding, function, stability, and association
have been studied in detail. Because the effect of excluded volume is more prominent in an extended state than in the native protein, a majority of these studies have been conducted in the unfolded state of different model proteins. Here, we have used fluorescence correlation spectroscopy (FCS) and other biophysical methods to investigate the effect of crowding agents Ficoll70 and Dextran70 on the nativelike state of cytochrome c from yeast. Yeast cytochrome c (y-cytc) contains a substantial expanded state in its native folded condition, which is present in equilibrium with a compact conformer in aqueous buffer. We have found that the crowding medium affects the native state equilibrium between compact and expanded states, shifting its population toward the compact conformer. As a result, the peroxidase activity of y-cytc decreases. Urea-induced protein stability measurements show
that the compaction destabilizes the protein due to charge repulsions between similar charged clusters. Interestingly, the time constant of conformational fluctuations between the compact and expanded conformers has been found to increase in the crowded milieu, suggesting a crucial role of the solution microviscosity
Incipient Twisted Ribbon Structure Stabilized by C12 Helical Turns in g4 /a Hybrid Peptide
No full textThe present work describes the synthesis and rystallographic analysis of ga hybrid peptides, Boc-g4
-L-Phe-L-Pro-NHMe (P1) and Boc-(g4 -L-Phe-L-Pro)2-NHMe (P2). Peptide P1 adopts twelve membered (C12) helical turn over ga segment, while P2 folds into twisted ribbon structure involving two expanded C12 helical turn
Assembly structure of proteins and antimicrobial peptides and their interaction with lipid bilayers
No full textProteins exist as functional monomers as well as in different multimeric units. Oligomerization is a chemical process that converts monomers to macromolecular complexes through assembly formation involving a finite degree of polymerization. Assembly structures vary from tetramer, hexamers, decamers etc. to oligomers, protofibrils and more complex fibrillar structures. Protein multimers and unidentified oligomers differ in several structural and/or functional aspects. Different attributes of the assembly formations of proteins constitute the central theme of my research and the thesis encompasses the observations related to the structural/conformational perturbation of the protein assemblies of different classes in the presence of different biomimetic environments. Assembly structures of proteins contribute to executing general functions of the cells and are often related to diseases. Antimicrobial peptides co-operatively accomplish functions whereas diseased proteins like beta amyloid peptides amalgamate to render diseased protein assemblies termed as oligomers/fibrils. Several other proteins like adenosine kinase (from Leishmania donovani), tend to aggregate naturally or selectively in presence of ADP, leading to inactivation. The self formed aggregates are of amorphous nature whereas the induced (in presence of ADP) aggregates are of amyloid nature. To propagate the idea of oligomerization in a specific direction emphasizing on the single fluorophore tyrosine, we aimed to study the conformational orientations of the Aβ peptides and purothionin (an AMP from wheat) in micellar environment and other biomimetic environments. The ordered aggregation or fibril formation in globular proteins either exists as oligomers in native state or due to the unfolding of the native state into an intermediate state which is amyloidogenic in nature. We have extended our research to study the sequence complexity of amyloidogenic regions/ aggregation prone regions in intrinsically disordered proteins from different databases (DisProt+IDEAL). Protein sequences are composed of amyloidogenic region(s) (AR) and low complexity region(s) (LCR), both of which play a significant role in protein aggregation and amyloid formation. We have investigated the sequence complexity of AR which is present in intrinsically disordered human proteins. Thus the investigation of the structural features of the assembly structures of proteins is critical in understanding the structural and functional aspects of proteins
Influence of Lipid Core Material on Physicochemical Characteristics of an Ursolic Acid-Loaded Nanostructured Lipid Carrier: An Attempt to Enhance Anticancer Activity
No full textThe impact of saturation and unsaturation in the fatty acyl
hydrocarbon chain on the physicochemical properties of nanostructured lipid
carriers (NLCs) was investigated to develop novel delivery systems loaded with
an anticancer drug, ursolic acid (UA). Aqueous NLC dispersions were prepared
by a high-pressure homogenization−ultrasonication technique with Tween 80 as
a stabilizer. Mutual miscibility of the components at the air−water interface was
assessed by surface pressure−area measurements, where attractive interactions
were recorded between the lipid mixtures and UA, irrespective of the extent of
saturation or unsaturation in fatty acyl chains. NLCs were characterized by
combined dynamic light scattering, transmission electron microscopy (TEM),
atomic force microscopy (AFM), differential scanning calorimetry, drug
encapsulation efficiency, drug payload, in vitro drug release, and in vitro
cytotoxicity studies. The saturated lipid-based NLCs were larger than unsaturated
lipids. TEM and AFM images revealed the spherical and smooth surface
morphology of NLCs. The encapsulation efficiency and drug payload were higher for unsaturated lipid blends. In vitro release
studies indicate that the nature of the lipid matrix affects both the rate and release pattern. All UA-loaded formulations exhibited
superior anticancer activity compared to that of free UA against human leukemic cell line K562 and melanoma cell line B1
Studies On Contribution Of Astrocytes To Neuron Death In Alzheimer’s Disease
No full textAstrocyte activation is one of the crucial characteristics of several neurodegenerative disorders including Alzheimer’s disease (AD). In AD, amyloid-β (Aβ) which is considered to be main causative factor for neuronal death, activates astrocytes. Astrocyte activation has both beneficial and detrimental effect on neuron health. But, neither the pathway through which astrocytes are being affected by Aβ nor the molecular mechanisms by which activated astrocytes are affecting neuron health has been deciphered. The endeavour of this thesis was first to characterise astrocyte activation upon Aβ exposure and identify the molecular mechanisms of astrocyte activation evoked by Aβ oligomer. Secondly our aim was to check how exactly astrocyte activation influence neuron health in a cell culture model of AD.
We found that a lower concentration (1.5μM) of Aβ oligomer causes astrocytes to proliferate and change their morphology whereas higher dose (4μM) of Aβ rather induces astrocyte cell death. Astrocyte cell death upon high Aβ concentration, then found to be mediated through FoxO3a/Bim/Caspase3 signal axis whereas a couple of MAPK pathways have been found to be involved in low Aβ dose. Interestingly we found a strong correlation between activation of both p38k and JNK/c-Jun pathways and Aβ induced astrocyte cell proliferation. Only p38K pathway was found to be involved in morphological changes of astrocytes due to Aβ.
Next in order to identify the role of astrocytes on neuron in AD condition we performed an investigation on the secretion profile of activated astrocytes. Tissue inhibitor of matrixmetalloproteinase-1 (Timp-1) which is a MMP inhibitor, has been found to be increased in Astrocyte conditioned medium (ACM) upon Aβ. AD brain contains high level of Timp-1 but its precise role on neurons remains to be elusive. Here we are demonstrating that Timp-1 is significantly neuroprotective against Aβ as recombinant Timp-1 rescued both cortical and hippocampal neurons but deprivation of timp-1 from ACM fails to protect neurons from Aβ. In addition the arborisations of neurons were poor particularly with the astrocytes in which timp-1 was knocked down in culture condition. Finally we found, Timp-1 restores Akt survival signal in neurons even in the presence of Aβ and thus can protect them
Functional assessment of tyrosinase variants identified in individuals with albinism is essential for unequivocal determination of genotype-to-phenotype correlation
No full textOculocutaneous albinism type 1 (OCA1), caused by pathogenic variations in the tyrosinase gene (TYR), is the most frequent and severe form of hypopigmentary disorder worldwide. While OCA1A manifests as a complete loss of melanin pigment, patients with OCA1B show residual pigmentation of the skin, hair and eyes. Limited experimental evidence suggests retention of TYR in
the endoplasmic reticulum (ER) causes OCA1 pathogenesis. However, a comprehensive functional analysis of TYR missense variations and correlation with genotype
is lacking. Objectives Functional characterization of nonsynonymous tyrosinase variants in patients with OCA1 reported in the Albinism Database, dbSNP and the published
literature, and an attempt to correlate them with reported and predicted phenotypes. Thirty-four reported missense variants of TYR were subcloned by sitedirected
mutagenesis, and the dual-enzyme activities of the variant proteins were compared with the wild-type. The degree of ER retention was also checked for each of the variants through endoglycosidase H (Endo H) digestion followed by
immunoprecipitation and densitometric analysis. Functional studies revealed one reported OCA1A variation with nearly100% enzyme activity, 10 OCA1B variants lacking any enzyme activity, eight nonsynonymous single-nucleotide polymorphisms (SNPs) with ~30–70% of enzyme activity, and three SNPs that completely lacked activity altogether. The
Endo H assay corroborated these results.Loss of enzyme activity of TYR variants was completely in agreement
with ER retention across all variants examined. The results of the assay clearly established that determination of the biological activity of identified variants in patients with OCA is essential to correlate the identified suspect genotype with the obvious phenotype of the disease
Role of Synthetic Metabolic Modulators in Targeting Mitochondrial Hyperpolarization to Repress Cancer Associated Hallmark Traits
No full textOne of the major health problems earmarked in today‘s world is cancer and its frequency of occurrence and the rate at which it causes fatality, poses to be a global
concern (1,2,3). A survey by World Health Organization (WHO) in the year 2008 suggested that grossly about 12.7 million people are affected with cancer and among
them about 7.6 million people have already died from this deadly disease worldwide. Furthermore, WHO predicts that this situation will worsen by 2030 where an
estimated 21.4 million new cases of cancer will arise, resulting in death of 13.2 million
cancer afflicted patients annually (4). The present day options of surgery, chemotherapy and radiotherapy would hardly suffice to tackle this magnanimous problem and therefore, newer and rational strategies are of utmost importance (5, 6).
Complementary and alternative medicines (CAMs) are already being considered for alleviating the problem of toxicity associated with conventional therapy and thus,
helping to provide a better life and longevity to cancer patients
Synthesis and Nanoformulation of Metabolic Modulators: A Strategic Approach towards Cancer Therapy
No full textThe process of drug discovery is at the interface of chemistry and biology and this work primarily focuses on the development of therapeutically important metabolic
modulators to combat cancer. Exclusive feature of the cancer cells include prominent metabolic imbalances that is preeminently aligned with the hallmarks of cancer. The
Warburg phenotype is a characteristic feature of the cancer cells that allows them a functional redundancy to enhance resistance towards apoptosis. Tumor cells undergo
glycolysis for ATP generation rather than utilizing TCA cycle and finally produce lactate in presence of lactate dehydrogenase A (LDH-A) that interconvert pyruvate to
lactate and is seen to be responsible for aggressive cancer outcomes. Therefore, LDH-A might be considered as a potential target in the arena of cancer therapeutics as blocking this enzyme would shut down energy supply and associated anabolic reactions. With this background information, this study was undertaken for the synthesis,
characterization and deciphering the tumoricidal potential of a novel “dual hit” molecule wherein it would compete with NADH and pyruvate to inhibit LDH-A and
selectively initiate apoptosis in the cancer cells, without toxic manifestations.
Another objective of the dissertation work was based on the concept of co-drug.Novel co-drug was derivatized and its PLGA based nanoparticles was conceived to be
developed that would further boost up the potential of the co-drug, highlighting the therapeutic prospectus of the metabolic modulator loaded nanoparticles in the rapidly expanding chapter of cancer therapeutics.
It is a well-established fact that two drugs when added in combination at defined doses can inhibit cancer in a synergistic way by changing the characteristic metabolic
signatures of cancer cells. Realizing the potential that derivatized metabolic modulators can destroy the cancer cells via two different routes, their combination at specific calculated doses was envisioned to mutually mitigate the drawbacks of standard chemotherapeutic regimens, concomitantly underlining the prospectus of metabolic modulators in the clinical setting
A rhodamine embedded bio-compatible smart molecule mimicking a combinatorial logic circuit and ‘key-pad lock’ memory device for defending against information risk
No full textOrganic molecules with the possibility of logic operations are highly useful building blocks for the development of molecule-based ‘‘intelligent’’ devices for information processing applications. We have designed herein a very simple bio-friendly chemosensor (LC) equipped with a rhodamine fluorophore moiety. This probe showed a chromo-fluorescence response profile for Al3+ but a colorimetric response for Cu2+ metal. The absorption responses of LC caused by these metal ions along with the ‘‘OFF–ON–
OFF’’ fluorescence behavior of an LC–Al3+ complex towards EDTA were employed for the development of a three-input and one output combinatorial molecular system. Interactions of the mentioned metal ions with LC in controlled sequential experiments gave fluorescence responses, enabling us to fabricate a ‘key-pad-logic’ function. So, a single molecular system performing such multiple ‘Boolean’ operations not only simplifies the complexity of a chemical driven ‘Intelligence’ device but also enriches the
security of such a device against information invasion due to the sequence controlled sensor–analyte interactions and may find potential applications in biocompatible molecular logic platform