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Bile dependent modulation of sensory transduction pathways in the enteric bacterium Vibrio cholerae
No full textVibrio cholerae is a Gram negative, non invasive enteric pathogen and the etiologic agent of human diarrheal disease, cholera. It colonizes the small intestine and secretes the major toxin CT responsible for the disease symptoms. In V. cholerae expression of CT and several other
virulence factors are coordinately controlled by a hierarchical regulatory cascade known as ToxR regulon. The cascade starts with AphA and AphB which are responsible for transcriptional activation of a transmembrane transcription factor TcpP, which together with another transmembrane DNA-binding protein ToxR, activates expression of the master regulator toxT. ToxT then promotes the next level of the virulence cascade by activating ctxAB and tcpA. During
infection V. cholerae encounters bile, which is a heterogeneous mixture of salts and acids and required for lipid emulsification in the intestine. Several studies have indicated that the bile exerts pleiotropic effects on V. cholerae that affects virulence, motility, biofilm formation etc. This dissertation reports that bile induces a LysR type transcriptional regulator LeuO in V. cholerae and LeuO regulates expression of ToxR regulon. In the present study a differential proteomic analysis and transcriptional study identified that, LeuO was induced in V. cholera classical biotype O395 in presence of bile in the stationary phase of growth. In a ΔleuO mutant
expression of the ctxA, tcpA genes and the master virulence regulator toxT were induced in presence of bile at stationary phase of growth, indicating that LeuO negatively regulates V. cholerae virulence factors production under these conditions. ChIP and EMSA analysis demonstrated that LeuO directly binds at toxT promoter and act as a negative virulence regulator in V. cholerae. As H-NS, the global repressor was known to negatively regulate toxT expression
in V. cholerae, role of H-NS and LeuO were studied. Both ChIP and EMSA experiments confirmed that in a ΔleuO mutant H-NS cannot bind at toxT promoter in presence of bile at
stationary phase of growth. LeuO cooperatively binds at toxT promoter, share overlapping binding sites with H-NS and thereby it helps H-NS to bind at this promoter to negatively regulate the gene expression. Previously LeuO was known as a global H-NS antagonist in other enteric bacteria like E.coli, Salmonella enteric etc. But this study demonstrates a novel function of LeuO, rather than
acting as an H-NS antagonist, LeuO facilitates binding of H-NS to the toxT promoter and thereby regulates V. cholerae virulence at stationary phase of growth in presence of bile
Elucidation of the DNA binding specificity of the natural plant alkaloid chelerythrine: A biophysical approach
No full textInteraction of the anticancer plant alkaloid chelerythrine with four sequence specific synthetic polynucleotides
was studied by spectroscopy and calorimetry experiments. The binding resulted in strong hypochromic and bathochromic effects in the absorption spectrum of the alkaloid, enhancement in the fluorescence with the AT polynucleotides and the homo-GC polynucleotide and quenching with the hetero- GC polynucleotide. Cooperative binding was observed with all the polynucleotides. Fluorescence polarization anisotropy, iodide quenching and viscosity results confirmed intercalative binding of the alkaloid. The binding resulted in the thermal stabilization of the polynucleotides and moderate perturbations in the B-conformation of the DNA. The high binding affinity values (�106 M�1) evaluated from the spectroscopic data was in excellent agreement with those obtained from calorimetry. The binding was exothermic and favoured by negative standard molar enthalpy and positive standard molar entropic contributions in all cases other than homo-AT polynucleotide, where it was endothermic and entropy driven.
Salt-dependent calorimetry data revealed that the binding reaction was driven mostly by non-polyelectrolytic
forces. The magnitude of the negative heat capacity values confirmed the role of significant hydrophobic effects in the interaction profile of the alkaloid with the polynucleotides. The results revealed the specificity of chelerythrine to follow homo-GC > hetero-GC > hetero-AT = homo-AT polynucleotid
Potential of the [99mTc(CO)3(H2O)3] Moiety for the Labeling of Biomolecules for Infection Diagnosis
No full textThe research work embodied in this thesis, which has been submitted to the Faculty of Engineering & Technology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, India for the award of degree of Doctor of Philosophy (Pharmacy) has been carried out at the CSIR-Indian Institute of Chemical Biology, Kolkata, under the supervision of Dr. (Mrs.) Mita Chatterjee Debnath, In-Charge Nuclear Medicine Division, Indian Institute of Chemical Biology (CSIR), Kolkata, India & Dr. Tuhinadri Sen, Associate Professor,Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. Chapter 1 begins with introduction; and a short review of literature. 99mTc(CO)3-ciprofloxacin prepared in high radiochemical yields with substantial stability and better infection imaging agent. These results encouraged us to radiolabel other fluoroquinolones with fac-[99mTc(CO)3(H2O)3] precursor. In an effort to understand the process of radiolabeling with fac-[99mTc(CO)3(H2O)3] precursor better, we have labeled a series of aminocarboxy ligands in Chapter 2. Thereafter we have successfully radiolabel fluoroquinolones in Chapter 3. Similarly thiosemicarbazone complexes with many metals show diverse chemical, physical and biological properties.(N) heterocyclic aldehydes and ketones posses a broad spectrum of potentially useful chemotherapeutic activities e. g. antibacterial, antiviral, antimalarial. The potentially useful biological properties of nitrofuryl thiosemicarbazones as well as outstanding importance of organometallic ion in radiopharmaceuticals chemistry encourage us to radiolabel thiosemicarbazones with 99mTc(CO)3-precursor in Chapter 4
Antimutagenic and anticancer activity of Darjeeling tea in multiple test systems
No full textDarjeeling tea, a most popular variety of black tea, though consumed by the people in different parts of world but its beneficial health effects have not been investigated in details. In this study, the antimutagenic and anticancer effect of Darjeeling tea extract (DTE) has been evaluated.
Antimutagenic activity of the DTE was carried out in two different strains of Salmonella typhimurium by
AMES test against a known mutagen benzo[a]pyrene (B[a]P) with S9 activation. Moreover, anticlastogenic property
of DTE was also measured by micronuclei formation (MN) against B[a]P with S9 activation in human lymphocytes.
The anticancer activity of the same was studied on U937 cell line. Here, Human PBMCs were used as the normal
cell control to identify selective anticancer activity of the extract against U937 cells. The results showed significant antimutagenic activity on bacterial strains. A significant decrease in MN was also observed in the DTE treated human lymphocyte cultures pretreated with B[a]P when compared with B[a]P treated cultures alone. The study clearly exhibited anticancer activity of the extract on U937 cell line. Further studies also revealed that apoptosis induction is an important mechanism behind the anticancer effect of DTE. Overall, this study indicates that DTE has significant antimutagenic and anticancer activities on bacterial and mammalian cells respectively
CSIR-IICB Research Productivity during 2001-2010: A Scientometric Analysis of Publications
Get PDFThe present research is an attempt to describe the quantity and quality of research publications of CSIR-IICB, Kolkata during ten years period from 2001-2010. The population under study was of 1228 documents on media literacy published through 2011. The results were analyzed based on date of publication, type of document, authorship pattern, source of publications, subject areas, affiliations, country origin of foreign collaborators. Citation indicators formed the second phase of investigation in the present study. There was increasing trend from 2001 to 2005 and reported 33.90% increase but there was considerable amount of fluctuation during 2006 to 2010. In the year 2006 53.01% increase was reported over 2001. The analysis of data based on document type indicated that, out of the 1228 publications, 1041 were articles (84.77%), 59 were reviews (4.80%), and 43 were conference papers (3.50%). It was disclosed from the analysis that major focus area of research as highest research papers were on Leishmania with 223 (29.89%) publications, followed by Synthesis of New Chemical Entities with 109 (14.61%) and Cancer Research with 101 (8.22%). The data analyzed to identify foreign collaboration and found the largest number of papers were published in Journal of Neurochemistry 27 (2.20%) followed by Tetrahedron Letters with 26 (2.12%) and Molecular and Cellular Biochemistry with 20 (1.63 %). The most cited articles from 2001 up to 2010 included 46 researches on the h-index. University of Calcutta was the top collaborator from the country followed by Jadavpur University and Chittaranjan National Cancer Institute
Critical stoichiometric ratio of CD4+ CD25+ FoxP3+ regulatory T cells and CD4+ CD25_ responder T cells influence immunosuppression in patients with B-cell acute lymphoblastic leukaemia
No full textRegulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to selfantigens. The frequency and suppressing activity of Treg cells in general
are high in different malignancies. We wanted to identify the role and regulation of CD4+ CD25+ FoxP3+ Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals
(n = 35). These diagnosed patients demonstrated a lower number of CD4+ CD25+ cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-b and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on
CD4+ CD25 responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4+ CD25+ FoxP3+ Treg cells became high with the progression of malignancy in B-ALL. Relative distribution
of Tresp and Treg cells was only ~5 : 1 in B-ALL but ~35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression
than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4+ CD25+ cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression.
Taken together, our study indicated that the CD4+ CD25+ FoxP3+ Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL
Antiviral activity of baicalin against influenza virus H1N1 pdm09 is due to modulation of NS1-mediated cellular innate immune responses
No full textBaicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo.Baicalin’s anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking.Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/ Akt signalling. Baicalin disrupted NS1–p85b binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39–43 of the NS1 RBD were
found to be crucial for the baicalin–NS1 interaction.
Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral
protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling
in cells
Studies on Proteins - Nucleic Acids Interactions in Regulation of Replication and Transcription
No full textFrom our knowledge of living species, it is quite hard to imagine a living organism without
macromolecules even at its most primitive stage of evolution. Macromolecules found within
the cell such as proteins and nucleic acids are the essence of life. Synthesis of all
biomolecules found within a cell depends on the collaboration of several protein molecules.
Within the cell, proteins are used as enzyme for catalysis, structural components and energy
generation. Proteins are produced through an amazing assembly process, involving DNA as a
template for three types of RNA (mRNA, rRNA and tRNA), which in turn act as different
components of protein synthesis. The central dogma of molecular biology (Fig. I.1) deals
with this detailed residue-by-residue sequential transfer of information (1). It also states that
information can never be transferred from a protein to another protein or nucleic acid. Each
step in this complicated synthesis is carried out by an enzyme, which, being a protein had to
be synthesized by the same process. In other words, the end products of this reaction aid in
the synthesis of the starting components and catalyze each reaction along the way, making up
a complicated series of interrelationships. In order to understand life, the appearance of this
entire machinery must be explained
Transient overexpression ofWerner protein rescues starvation induced autophagy inWerner syndrome cells
No full textReduced autophagy may be associated with normal and pathological aging. Here we report a link between
autophagy and Werner protein (WRNp), mutated in Werner syndrome, the human premature aging Werner
syndrome (WS).WRNmutant fibroblast AG11395 and AG05229 respondweakly to starvation induced autophagy
compared to normal cells. While the fusion of phagosomes with lysosome is normal, WS cells contain fewer
autophagy vacuoles. Cellular starvation autophagy in WS cells is restored after transfection with full length
WRN. Further, siRNA mediated silencing of WRN in the normal fibroblast cell line WI-38 results in decreased
autophagy and altered expression of autophagy related proteins. Thus, our observations suggest that WRN may
have a role in controlling autophagy and hereby cellular maintenance
Probing the structure of Mycobacterium tuberculosis MbtA: model validation using molecular dynamics simulations and docking studies
No full textMultidrug resistance capacity of Mycobacterium tuberculosis demands urgent need for developing new antitubercular
drugs. The present work is on M. tuberculosis-MbtA, an enzyme involved in the biosynthesis of siderophores, having a
critical role in bacterial growth and virulence. The molecular models of both holo and apo forms of M. tuberculosis-
MbtA have been constructed and validated. A docking study with a series of 42 5′-O-[N-(salicyl) sulfamoyl] adenosine
derivatives, using GOLD software, revealed significant correlation (R2 = 0.8611) between Goldscore and the reported
binding affinity data. Further, binding energies of the docked poses were calculated and compared with the observed
binding affinities (R2 = 0.901). All-atom molecular dynamics simulation was performed for apo form, holo form without
ligand and holo form with ligands. The holo form without ligand on molecular dynamics simulation for 20 ns converged
to the apo form and the apo form upon induced fit docking of the natural substrate, 2,3-dihydroxybenzoic acid-adenylate,
yielded the holo structure. The molecular dynamics simulation of the holo form with ligands across the time period of
20 ns provided with the insights into ligand–receptor interactions for inhibition of the enzyme. A thorough study
involving interaction energy calculation between th ligands and the active site residues of MbtA model identified the key residues implicated in ligand binding. The holo model was capable to differentiate active compounds from decoys. In the absence of experimental structure of MbtA, the homology models together with the insights gained from this study will promote the rational design of potent and selective MbtA inhibitors as antitubercular therapeutics