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Cancer Cell Specific Delivery of Photosystem I Through Integrin Targeted Liposome Shows Significant Anticancer Activity
No full textMany anticancer drugs are developed for the treatment of cancer from natural sources. Photosystem I (PSI), a protein complex present in the chloroplast, is involved in
photosynthesis and generates reactive oxygen species (ROS)
in plant. Here, we used the ROS generation property of PSI
for cancer therapy. We show that PSI can enter into different kinds of cancer cell like human lung carcinoma (A549) and mouse melanoma (B16F10) cell lines and generate ROS inside the cells. It inhibits the proliferation of cancer cell and causes apoptotic death of cancer cells. We also show that PSI induces apoptosis through mitochondria-dependent internal pathway, induces caspase3, causes DNA fragmentation, and arrests cell cycle at SubG0 phase. We also prepared, using C16-LDV lipopeptide [C16 long chain attached on the N-terminal of the tripeptide containing amino acids leucine (L), aspartic acid (D), and valine (V) abbreviated as NH2-LDV-COOH], α4β1 integrin targeted liposomal formulation of PSI, which specifically kills the cancer cell without affecting normal cells, and it is found to be more potent compared to clinically used drug doxorubicin. Finally, we found that LDV liposomal formulation of PSI inhibits the growth of tumor in C57BL/6J mice model
Attenuation of Helicobacter pylori-induced gastric inflammation by prior cag− strain (AM1) infection in C57BL/6 mice
Full text linkHelicobacter pylori, colonize in stomach of ~50% of the world population. cag pathogenicity Island of H.
pylori is one of the important virulent factors that attributed to gastric inflammation. Coinfection with H. pylori strain with different genetic makeup alters the degree of pathogenicity and susceptibility towards antibiotics. The present study investigates host immunomodulatory effects of H. pylori infection by both cag+ strain (SS1) and cag− strain (AM1). C57BL/6 mice were infected with AM1 or SS1 strain as well as AM1 followed by SS1 (AM1/SS1) and vice versa.
Results: Mice infected with AM1/SS1 strain exhibited less gastric inflammation and reduced proMMP9 and proMMP3
activities in gastric tissues as compared to SS1/SS1 and SS1/AM1 infected groups. The expression of both MMP9 and
MMP3 followed similar trend like activity in infected tissues. Both Th1 and Th17 responses were induced by SS1 strain more profoundly than AM1 strain infection which induced solely Th1 response in spleen and gastric tissues. Moreover, IFN-γ, TNF-α, IL-1β and IL-12 were significantly downregulated in mice spleen and gastric tissues infected by AM1/SS1 compared to SS1/SS1 but not with SS1/AM1 coinfection. Surprisingly, IL-17 level was dampened significantly in AM1/ SS1 compared to SS1/AM1 coinfected groups. Furthermore, number of Foxp3+ T-regulatory (Treg) cells and immunosuppressive
cytokines like IL-10 and TGF-β were reduced in AM1/SS1 compared to SS1/SS1 and SS1/AM1 coinfected
mice gastric tissues.
Conclusions: These data suggested that prior H. pylori cag− strain infection attenuated the severity of gastric pathology induced by subsequent cag+ strain in C57BL/6 mice. Prior AM1 infection induced Th1 cytokine IFN-γ, which reduced the Th17 response induced by subsequent SS1 infection. The reduced gastritis in AM1/SS1-infected mice
might also be due to enrichment of AM1- primed Treg cells in the gastric compartment which inhibit Th1 and Th17
responses to subsequent SS1 infection. In summary, prior infection by non-virulent H. pylori strain (AM1) causes reduction of subsequent virulent strain (SS1) infection by regulation of inflammatory cytokines and MMPs expressio
Cdc25A phosphatase: a key cell cycle protein that regulates neuron death in disease and development
Full text linkCell cycle molecules are mostly dormant in differentiated
neurons that are post-mitotic and in the G0 state of the cell cycle. However, a wealth of evidence strongly suggests that in response to a wide variety of apoptotic stimuli, including trophic factor deprivation, exposure to β-amyloid (Aβ) and DNA damage, neurons emerge from theG0 state with aberrant expression/activation of cell cycle proteins.1 This emergence is characterized by a consistent set of events related to the cell cycle that culminate in neuron death. Initial responses include
activation of G1/S cyclin-dependent kinases (Cdks), such as Cdk4 that in turn phosphorylate retinoblastoma (pRb) family proteins and lead to dissociation of repressor complexes comprising E2F and pRb proteins, so that E2F-binding genes are de-repressed. Among genes that are de-repressed by loss of E2F-Rb family complexes are the B- and C-myb transcription factors that in turn transactivate Bim, a pro-apoptotic protein that promotes caspase activation and subsequent neuron death.1–4 This set of events has been termed the ‘apoptotic cell cycle pathway’.Cell division cycle 25A (Cdc25A), a member of a family comprising Cdc25A, B and C, is a dual specificity phosphatase that dephosphorylates inhibitory phosphates on adjacent threonine and tyrosine residues of Cdks such as Cdk4.5 This step is essential for initiation of cell cycle in proliferating cells. However, it was not known whether in the non-dividing neurons, the same events would activate the apoptotic cell cycle pathway. In our recent paper published in Cell Death Discovery,6 we report several novel findings regarding the potential role of Cdc25A in neuron death. First, Cdc25A is
required for activation of the apoptotic cell cycle pathway and neuron death in response to nerve growth factor (NGF) deprivation and Aβ treatment. Second, Cdc25A acts upstream of Cdk-mediated Rb phosphorylation and caspase-3 cleavage. Third, NGF deprivation and Aβ lead to rapid increases in Cdc25A mRNA and protein levels. NGF withdrawal causes an increase in Cdc25A activity as well. These events occur at about the same time that apoptotic insults lead to Cdk4 activation and Rb phosphorylation in our experimental systems and well precede evident signs of neuron death
Role of Fat Metabolism in Cellular Differentiation
No full textThe metabolic phenotype of a cell can change in response to substrate availability and the metabolic demands of proliferation, growth and cell survival. The adaptation of the cellular metabolism to suit the developmental stages or during unrestricted proliferation is known as
metabolic remodelling. The major shift in the metabolism occurs between the choices of the fuel; accordingly the cell’s metabolic phenotype could either be preferentially glycolytic or oxidative. Cellular differentiation entails a shift in the metabolic phenotype as the energy
demands and requisites for raw materials reduce once the cells are committed to a specific lineage. Previous studies have speculated a shift towards oxidative phosphorylation as the major metabolic phenotype in the differentiated cells. Adipose triglyceride lipase (ATGL) is the ratelimiting enzyme for triacylglycerol (TG) catabolism and is considered to be a key driver of the
oxidative phosphorylation (OXPHOS). However, the role of fat metabolism has been relatively unexplored in the context of metabolic switching during differentiation. This study thus aimed to unravel the role of ATGL catalyzed lipolysis in metabolic rewiring during differentiation. The study was further expanded to investigate the effect of the ATGL in driving the skeletal muscle mitochondrial metabolism and its implication in muscle frailty or sarcopenia. Initially, several in vitro models of differentiation of the mesenchymal lineage were screened for the metabolic shift towards an oxidative phenotype and it was found to be dependent on ATGL.Mouse placental differentiation was chosen for further studies and ATGL induction was found to be concomitant with increased fatty acid oxidation (FAO) and oxygen consumption rates (OCR) and paralleled the PPARγ expression patterns; although the key glycolytic enzyme, hexokinase II and the lactate levels were not significantly repressed suggesting that glycolysis is not completely dispensable.Next, the role of ATGL mediated lipid metabolism was investigated in another cellular differentiation system, the skeletal muscle. Myogenesis, marked by the fusion of myoblasts into myotubes, parallels mitochondrial biogenesis which is regulated via a transcriptional program driven by PPARα and its coactivator PPAR-γ coactivator-1α (PGC1α). However, the role of ATGL dependent lipolysis in skeletal muscle mitochondrial metabolism was poorly understood.
ATGL was exclusively induced during differentiation and it was demonstrated by genetic as well as pharmacological interventions in both in vitro and in vivo systems, to be a transcriptional target of PPARα. However, augmented expression of ATGL did not alter the effects of PPARα
or its agonist fenofibrate dependent lipid metabolism and insulin sensitivity. Both in vitro and in vivo, ectopic expression of ATGL significantly enhanced while depletion of ATGL attenuated mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) without alteration in mitochondrial content. Over expression of ATGL rendered PPARα and PGC1α
redundant and they are unable to function upon depletion of ATGL. Further, in middle aged rats, fenofibrate induced ATGL expression selectively in the oxidative soleus muscle, where PPARα- ATGL pathway sustained mitochondrial oxidative function. In conclusion, ATGL driven lipid catabolism is instrumental in driving the oxidative phosphorylation during cellular differentiation and is indispensable for the function of the PPARa mediated mitochondrial oxidative program in the skeletal muscle
Vesicular delivery of biologically active compounds for combating hepatocellular mitochondrial oxidative damage
No full textLiver, the largest organ in our body, metabolizes different chemicals and xenobiotic substances like drugs, carcinogens etc. that enter into the body and therefore become prone to these chemical mediated oxidative damage. According to World Health Organization (WHO), right now there are around 5.5 million chronic liver disease patients now in USA and if considered globally the number is much bigger. Different xenobiotic materials that cause serious health hazards are alcohol, carbon tetrachloride (CCl4), aspirin, arsenic, diethylnitrosamine (DEN) etc.
Since human liver metabolizes CCl4 in a manner similar to that of rodents, CCl4 induced liver injury can be an appropriate model for chemical induced liver injury study. Short term administration of CCl4 causes hepatic injury, centrolobular necrosis, steatosis and inflammation. Oxidative stress is a principal aspect of hepatic damage. The mitochondrion is the bioenergetic and metabolic center of eukaryotic cells. Mitochondrion is the target as well as the source of ROS generated through CCl4 administration. Our studies showed that apart from the formation of mitochondrial ROS CCl4 also damages mitochondria and triggers mitochondria mediated apoptotic pathway. Curcumin, although has anti-oxidative, anti-carcinogenic, anti-inflammatory and immunomodulatory properties, but its low aqueous solubility and poor gastrointestinal absorption has limited its therapeutic application. Our study demonstrated that both liposomal and nanoparticulated formulation can increase the bioavailability of free curcumin, still nanoparticulated formulation has a relative bioavailability four times higher than liposomal formulation. It was also found that both liposomal as well as nanoparticulated formulations can increase the efficacy of curcumin many folds. However, due to its small size, better cellular absorption and longer persistence in the circulating system, curcumin loaded nanoparticles showed better protection from oxidative stress, mitochondrial damage and disruption from cellular architecture and thus can be considered as a promising therapeutic strategy against liver toxicity
Folding And Conformation Studies Of Therapeutically Rel Kunduevant Proteins Using Biophysical And Biochemical Methods
No full textlandscape to achieve the correct physical conformation at a minimal energy state. A series of interactions between different amino acid residues in a regulated manner
ultimately lead to a stable conformation. However, proteins sometimes fail to maintain their correct folded state under some circumstances. Misfolded or
aggregated proteins are found to be associated with different diseases.In this study, we studied different conformational switches in the folding pathway
of proteins that can lead to correct folding or misfolding. We chose three model
systems of different proteins. These proteins have different secondary and tertiary
structures in their native forms. Interestingly, all these three proteins are shown to be implicated in pathogenesis of three different diseases.
In this thesis, we investigated the equilibrium unfolding transition of a Mycobacterium protein MPT63. MPT63 has been shown to raise humoral responses
in tuberculosis patients. Our study showed that in spite of being a complete β-sheet protein, MPT63 has a prominent propensity towards helix structures in its early intermediates. Far UV-CD and FTIR spectra suggested that the low pH intermediate of MTP63 has enhanced helical contents, while fluorescence correlation
spectroscopy suggested a significant compaction. Furthermore, molecular dynamicssimulation complemented the experimental results. Our data suggested that the
secondary structure preferences of the local interactions in early folding pathway of a protein might not always follow the native conformation. Moreover, we showed
that this non-native intermediate has a propensity to form amyloid aggregates. Our next model protein system was alpha-synuclein which is the major component
in the amyloid plaques found in Parkinson’s disease. We used spectroscopic measurements at ensemble and single molecule resolution to study how the late and
early events of -syn aggregation modulate each other. At the early stage, the protein fluctuated between conformers of different hydrodynamic radii and forms
oligomers. The late stage followed through the formation of different sized iv intermediate species eventually generating amyloid fibrils. Additionally, we used
two small molecules and investigated their effect on -syn folding/aggregation landscape. We chose an inhibitor arginine, and a facilitator glutamate based on their
respective abilities to delay or accelerate the late stage of aggregation (amyloidosis) in solution and in live neuroblastoma cell line (SH-SY5Y). In the presence of the inhibitor molecule, the formation of a compact conformer was favored, while the addition of the facilitator sped up oligomerization. Our data also suggested that when arginine and glutamate were present together in a solution, they cancelled each
other’s effects. Our last model system was a Saccharomyces serevisiae protein Sup35. Sup35 is the
yeast version of translation termination factor. Incorrect folding due to mutation or other external triggers lead to Sup35 aggregation. Aggregated Sup35 showed human dentified and characterized the aggregation of the GFP-tagged Sup35 inside yeast cells. In this
study, we chose a small molecule trehalose and investigated its effect on the Sup35 aggregation. Trehalose is the most studied chemical osmolyte which is naturally synthesized in yeast and reported to protect proteins from misfolding in stressed conditions. Additionally, we investigated the effect of different heatshock treatments on Sup35 expression pattern and aggregation inside live yeast cells in the absence
and presence of trehalose. Our studies suggested that trehalose is able to successfully prevent the heatshock-induced enhanced aggregation of Sup35 in live
yeast cells
Dual histone reader ZMYND8 inhibits cancer cell invasion by positively regulating epithelial genes
No full textEnhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasionassociated genes through local epigenetic modifications which are recognized by a class
of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has
recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential
gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by
ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration
assays, its anti-invasive nature is evident. Interestingly, epithelial–mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/ H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function
Understanding the molecular mechanisms of induction of oxidative stress and insulin resistance due to the treatment of free fatty acid and high fat diet in vitro and in vivo models
No full textObesity has become the predominant health burden affecting over a third of the world’s population today. Obesity is a complicated multifactorial and
multi organ-centric disease that has been fueled by economic growth, sedentary lifestyle, urbanization and nutritional adaptation to calorie rich diets and processed food. Several complex metabolic diseases of heterogeneous etiology are associated
with obesity; type 2 diabetes mellitus (T2DM) is one of such predominant disorders.
Metabolic hallmark of T2DM is insulin resistance, the proposed underlying link between sedentary lifestyle and metabolic syndrome. The understanding of its
complex etiology and a proper detailed overview of its perplexing mechanisms can pave the way towards its therapeutics. As the pathophysiology of T2DM apart from
pancreatic β-cells, mainly surrounds liver and peripheral insulin target tissues i.e.,skeletal muscle, the current study has been focused on deciphering the metabolic
mechanisms in these organs. Moreover, increased oxidative stress has always appreciated as an inimical factor leading to insulin resistance in these tissues. The
present investigation has been designed to study metabolic contrivances behind reactive oxygen species generation during insulin resistance
CELL SIGNALING AND IMMUNE RESPONSE DURING LEISHMANIA DONOVANI INFECTION OF HOST MACROPHAGE
No full textExploring The Genome And Proteome Landscapes Of The Genus Bacillus In Quest Of Lineage And Niche Specific Traits Through In Silico Analysis
No full textMicrobes are one of the earliest organisms inhabiting earth since times life has originatedon the surface of this planet about 3.5 billion years ago. During these 3.5 billion years, microbes have evolve in a way to adapt themselves to all possible environment, even to
the most extreme and adverse ones – thanks to the process of molecular evolution. Any study on evolution of life will be incomprehensive without studying the molecular
evolution of microbes - the vehicles of all major evolutionary processes till to this date. With the advent of nextgen sequencing technologies, microbial sequences are regularly coming up at a break neck speed and this, in turn, has boosted up the studies on molecular evolution in microbes from diverse lineages and of widely varying life-style. As revealed in these studies, the genome composition of microbes, especially of bacterial species, often bear the signatures not only of their phylogenetic positions, but also of their niches and/or life-styles. The genetic constitution of a bacterium normally reflects a complex
interplay between its taxonomic legacy and ecological constraints. While the legacy of the ancestral genetic trends is, by and large, followed within a specific lineage, adaptation to distinct niches or life-styles may cause fine-tuned variations in gene/protein composition across its members, even across the strains of a single species. In an attempt to probe into the process of such fine-tuned optimization between the taxonomic legacy
and ecological adaptation, the present study was designed to focus on molecular evolution in a specific bacterial lineage – the Firmicutes with a special reference to its genera Bacillu