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Biophysical studies on the interaction of the alkaloid chelerythrine with nucleic acids
No full textDNA-deoxyribonucleic acid is the blueprint for life. It is present in organisms ranging from
the smallest bacterium to the largest whale. DNA carries most of the genetic instructions
used in the development, functioning and reproduction of well known living organisms.
James Dewey Watson and Francis Harry Compton Crick had revolutionized the field of
molecular biology and medicine by proposing the structure of deoxyribonucleic acid through
model building studies in their celebrated paper published in the Nature magazine of April
23 (Watson and Crick, 1953). Although the Watson and Crick was known as father of DNA the
preliminary research on DNA was started many decades ago on 1868 by Swiss chemist
Friedrich Miescher. Miescher in 1868 detected a phosphorus-containing substance from the
nuclei of pus cells obtained from discarded surgical bandages. He named it ‘nuclein’ consisting
of an acidic portion which we know today as DNA. In 1878, Albrecht Kossel isolated the nonprotein
component of “nuclein”, the nucleic acid, and later isolated its five primary
nucleobases (Albrect, 1879). In 1919, Phoebus Levene identified the base, sugar and phosphate
nucleotide unit (Levene, 1919). Levene suggested that DNA consisted of a string of nucleotide
units linked together through the phosphate groups. Levene thought the chain was short and
the bases repeated in a fixed order. In 1937, William Astbury produced the first X-ray diffraction
pattern that showed that DNA had a regular structure (Astbury and Florence, 1938). In 1944
Oswald Avery and his coworkers discovered that DNA carries a cell’s genetic material and
can be altered through transformatio
Studies Of Cytochrome c Folding And Conformational Dynamics Using Experimental And Computational Methods
No full textProtein folds from an unstructured/unfolded state to a more organized compact structure in order to attain a functional form. The conformational search during the folding process is difficult to comprehend as the time taken by a polypeptide chain, to search the infinite number of possible conformations, is surprisingly fast. It has been found that, this complex search algorithm is governed by multiple parameters, which include; amino acid sequence, structural architecture, solvent conditions, interactions with other macromolecules like proteins or protein complexes and the presence of chaperones etc. It has been found that the folding energy landscape of a protein is like a funnel having rough surface, containing various kinetic traps. Depending upon the above mentioned parameters, the protein chain samples various minima in the folding energy landscape, while rolling down the funnel surface. The conformations at each minimum have varying degree of stability and in general, the functional form of a protein is relatively more stable as compared to others states, like intermediate states. It is to be noted that a single protein can attain various functional forms and these functional forms are not necessarily the most stable state of the protein. The most stable state of a protein is the aggregated state, and this end state can be achieved by the protein, in various routes. The normal or favored route is highly populated by functional forms and on-pathway intermediates help the folding flux towards the functional form. The structural degradation over time of the functional form, in-vitro, leads to the aggregation while in-vivo the degraded and nonfunctional forms can be taken care-off by protease actions. Nevertheless, the folding process is not error free; and peptide chain folding can be derailed from the normal route to form miss-folded species / off-pathway intermediates and ultimately leading to aggregation.
The overall folding process can be complex with respect to both execution mechanism and outcome possibilities. It has been found that the folding energy landscape can be complemented by binding energy landscape to generate stable states, which may have functional as well as non-functional aspects. The conformational heterogeneity and the associated dynamic variability of a protein contribute significantly to define its folding, ligand binding and aggregation landscape.
In this thesis, we have investigated the conformation and dynamic heterogeneity of a protein using cytochrome c as a model. To do this, we have studied the binding of cytochrome c, from higher eukaryote (horse) and lower eukaryote (yeast), with negatively charged phospholipid, cardiolipin. This investigation may have important implications in the context of the understanding of the secondary functions of cytochrome c, which become important during apoptosis. We have found that the secondary function of cytochrome c, specially the pro-apoptotic activity, varies among the source of cytochrome c. We have used fluorescence correlation spectroscopy to find out the presence of three separate conformers, having varying degree of brightness (a dark compact conformer, a bright expanded conformer and oligomers), which are in equilibrium with each other in the presence of cardiolipin. An understanding of the complex interplay within these three populations is crucial for the pro-apoptotic functions of these proteins. We found that cytochrome c from yeast fluctuates more compared to the cytochrome c from horse. As a result of this fluctuation, the yeast protein is peroxidase active in the native state, but horse cytochrome c requires the help of cardiolipin to become peroxidase active. In the presence of cardiolipin, cytochrome c from yeast becomes aggregation prone and its peroxidase activity decreases. We have concluded that, in the case of yeast cytochrome c the formation of non-functional aggregates could be responsible for its lack of pro-apoptotic activity. We have also identified the key residues that may be responsible for higher degree of fluctuations in yeast cytochrome c. Subsequently, we have explored the folding as well as cardiolipin binding landscape of yeast cytochrome c in synthetic crowded media. We have found that the presence of crowded media disfavors the expanded state populations of yeast cytochrome c. This behavior helps the protein to control the unwanted peroxidase activity inside a healthy yeast cell. Our study also shows that the favored compactness in yeast cytochrome c population destabilizes the protein. We have additionally reported that although the crowded media favors the compact conformer due to exclusion volume effect, the sub-folding rate between expanded state and compact state is affected by the micro-viscosity of the crowded media. In a separate work we have established that fluorescence correlation spectroscopy and molecular dynamic simulation can be used as a complementary technique to explore the unfolding of a truncated cytochrome c from bacteria (Pseudomonas aeruginosa) (cytochrome VIII
c551). By using these techniques, we have characterized the intermediate state of cytochrome c551 demonstrating the shape change of this protein, which occurs as a result of unfolding
Sequencing,De novo Assembly, Functional Annotation and Analysis of Phyllanthus amarus Leaf Transcriptome Using the Illumina Platform
Get PDFPhyllanthus amarus Schum.and Thonn. a widely distributed annual medicinal herb has a long history of use in the traditional system of medicine for over 2000 years. However, the lack of genomic data for P.amarus,a non-model organism hinders research at the molecular level. In the present study ,high-through put sequencing technology has been employed to enhance better under standing of this herb and provide comprehensive genomic in formation foruture work
Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages
Get PDFPromastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW
264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain preexposed
macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in futur
BPGA- an ultra-fast pan-genome analysis pipeline
Get PDFRecent advances in ultra-high-throughput sequencing technology and metagenomics have led to a
paradigm shift in microbial genomics from few genome comparisons to large-scale pan-genome studies
at different scales of phylogenetic resolution. Pan-genome studies provide a framework for estimating
the genomic diversity of the dataset, determining core (conserved), accessory (dispensable) and
unique (strain-specific) gene pool of a species, tracing horizontal gene-flux across strains and providing
insight into species evolution. The existing pan genome software tools suffer from various limitations
like limited datasets, difficult installation/requirements, inadequate functional features etc. Here we
present an ultra-fast computational pipeline BPGA (Bacterial Pan Genome Analysis tool) with seven
functional modules. In addition to the routine pan genome analyses, BPGA introduces a number of
novel features for downstream analyses like core/pan/MLST (Multi Locus Sequence Typing) phylogeny,
exclusive presence/absence of genes in specific strains, subset analysis, atypical G + C content analysis
and KEGG & COG mapping of core, accessory and unique genes. Other notable features include
minimum running prerequisites, freedom to select the gene clustering method, ultra-fast execution,
user friendly command line interface and high-quality graphics outputs. The performance of BPGA has
been evaluated using a dataset of complete genome sequences of 28 Streptococcus pyogenes strains
One-Pot Synthesis and Evaluation of Antileishmanial Activities of Functionalized S‑Alkyl/Aryl Benzothiazole-2-carbothioate Scaffold
No full textThe synthesis of hitherto unreported S-alkyl/
aryl benzothiazole-2-carbothioate is reported from thiols,
oxalyl chloride, and 2-aminothiophenols using 10 mol % ntetrabutylammonium iodide (TBAI) as catalyst in acetonitrile through multicomponent reaction (MCR) strategy. The present protocol favored formation of benzothiazoles and thioesters via simultaneous formation of C−N and C−S bonds in good yields with a wide range of substrates. A few of the synthesized derivatives were evaluated for their antimicrobial activity against the protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Further, these
compounds displayed no toxicity toward macrophage RAW
264.7 cells and are therefore nontoxic and effective antileishmanial leads. In silico docking studies were performed to understand the possible binding site interaction with trypanothione reductase (TryR)
Studies on Functional Role of Docosahexaenoic acid (DHA) in the Brain
No full textDocosahexaenoic acid (DHA), an important ω-3 fatty acid, showed opposite effect in two different types of cells, inducing apoptosis in neoplastic cells but promoting differentiation in normal cells. With a view to understand more about the mechanisms of such divergent action
of DHA, the present study was conceived. Treatment of C6 glioma and SH-SY5Y cell lines, representing the neoplastic cells, with 100 μM DHA for 24 h caused significant increase in apoptosis, as determined by TUNEL staining, whereas primary astrocytes cultures, representing normal healthy cells were unaffected. Employing proteomic approach, we have
identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 and glutathione–S-transferase P1 subunit showed upregulation by DHA in the cancer cells. Western blot analysis also identified upregulation of PPARα and the MAP kinases, ERK, JNK and p38 as well as increased ROS production selectively in the cell lines. Together, activation of multiple apoptotic pathways in
association with excess ROS and activated MAPKs appear to promote cancer cell death by DHA. Like thyroid hormones (TH), DHA also facilitated astrocytes differentiation in culture, involving a downstream role β-adrenergic receptor (β-AR) system. We explored the detailed signaling mechanisms during the differentiation process induced by TH or DHA. TH, caused an immediate decrease in the specific binding of 125I-pindolol to β2-AR in cell membranes
with a concomitant increase in β2-AR levels in the cytosol, which could be blocked by endocytic inhibitors, suggesting endocytosis of β2-AR. qRT-PCR and western blot analysis
together with knockdown and overexpression experiment demonstrated that β-arrestin-1 is transcriptionally upregulated by TH facilitating increased endocytosis of β2-AR, required for endosomal ERK activation to drive the differentiation process. DHA also promoted β2-AR
internalization and its subsequent down-stream events to induce differentiation of the cells, not by increasing the expression of β-arrestin-1 but by increasing the expression of β2-AR levels
A Conserved Helicobacter pylori Gene, HP0102, Is Induced Upon Contact With Gastric Cells and Has Multiple Roles in Pathogenicity
No full textContact with host cells is recognized as a signal capable of triggering expression of bacterial genes important for host pathogen interaction.Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly upregulated expression of a gene, HP0102,in the adhered bacteria in all strains examined, including several Indian clinical isolates. The gene is highly conserved and ubiquitously present in all 69 sequenced H. pylori genomes at the same genomic locus, as well as in 15 Indian clinical isolates. The gene is associated with 2 distinct phenotypes related to pathogenicity. In AGS cell–adhered H. pylori, it has a role in upregulation of cagA expression from a specific σ28-RNAP promoter and consequent induction of the hummingbird phenotype in the infected AGS cells.
Furthermore, HP0102 has a role in chemotaxis and a ΔHP0102 mutant exhibited low acid-escape response that might account for the poor colonization efficiency of the mutan
Influence of single and binary doping of strontium and lithium on in vivo biological properties of bioactive glass scaffolds
Get PDFEffects of strontium and lithium ion doping on the biological properties of bioactive glass (BAG) porous
scaffolds have been checked in vitro and in vivo. BAG scaffolds were prepared by conventional glass melting route and subsequently, scaffolds were produced by evaporation of fugitive pore formers. After thorough physico-chemical and in vitro cell characterization, scaffolds were used for pre-clinical study. Soft and hard tissue formation in a rabbit femoral defect model after 2 and 4 months, were assessed using different tools. Histological observations showed excellent osseous tissue formation in Sr and Li + Sr scaffolds and moderate bone regeneration in Li scaffolds. Fluorochrome labeling studies showed wide regions of new bone formation in Sr and Li + Sr doped samples as compared to Li doped samples. SEM revealed abundant collagenous network and minimal or no interfacial gap between bone
and implant in Sr and Li + Sr doped samples compared to Li doped samples. Micro CT of Li + Sr samples showed highest degree of peripheral cancellous tissue formation on periphery and cortical tissues inside implanted samples and vascularity among four compositions. Our findings suggest that addition of Sr and/or Li alters physico-chemical properties of BAG and promotes early stage in vivo osseointegration and bone remodeling that may offer new insight in bone tissue engineering
Structural and functional characterization of type three secretion associated proteins from Yersinia enterocolitica and vitamin biosynthesis pathway proteins from Pseudomonas aeruginosa
No full textNumerous gram negative bacteria employ contact dependent Type Three Secretion System
(T3SS) to inject virulence proteins into their host. Species like Yersinia, Pseudomonas, Shigella,
Xanthomonas, Bordetella, Erwinia, Escherechia coli can infect human, animals and plants causing a
number of diseases. T3SS uses a multiprotein complex structure (‘injectisome’) embedded into bacterial
cell to inject toxins which targets different signaling cascade in their respective hosts. T3SS consists of
different types of proteins : apparatus proteins- involved in architecture of injectisome; translocatorssits
on top of the injectisome to create a protein into host cell membrane; effectors-virulence toxins
chaperones specific for effectors/translocators and proteins involved in regulation, i.e. regulators.
Yersinia enterocolitica is an enteropathogenic bacteria which causes gastrointestinal infection
including ‘yersiniosis'. In immunocompromised adults and children, systemic infection by this
bacteria leads to high mortality rates. In addition to the plasmid encoded Ysc-yop T3SS, a
genomic encoded 200kb YSA-PI region T3SS also exists in Yersinia enterocolitica, called ysaysp
system. Apart from this region there are also some T3SS genes scattered across the geno of Yersinia enterocolitica. YsaN is a putative ATPase in this genomic encoded T3SS as inferred from sequence homology. Pseudomonas aeruginosa is an opportunistic pathogen associated with diseases like pneumonia, urinary tract infection, nosocomial infections etc in immunocompromised individuals, pateients with burns and wounds. So far very little medication is available to kill this organism. Microorganism and plants are capable of producing their own vitamins using specific pathways catalysed by certain enzymes. However humans usually depend on dietary source to assimilate vitamins. Hence these unique pathways can be used as potential drug targets for complete eradication of such organism. Pantothenate and Coenzyme A biosynthesis pathway is a significant pathway in this regard. Pantothenate is an important component of CoA required in central metabolism like fatty acid synthesis as well as in synthesis of polyketides. CoA is synthesized from pantothenate in five steps and the penultimate step is catalyzed by phosphopantethiene adenylyltransferase (PPAT), a product of coaD gene