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A potent bidirectional promoter from the monocot cereal Eleusine coracana
No full textRagi bifunctional α-amylase-trypsin inhibitor (RBI) of Eleusine coracana (L.) Gaertn. (finger millet) simultaneously inhibits α-amylase and trypsin. In continuation of previous work on the cloning, expression and characterization of RBI, a bidirectional promoter from finger millet was explored on the basis of experimental observations. Two trypsin inhibitors were identified while purifying RBI from a trypsin-Sepharose column eluent. Using an FPLC gel filtration column, these three inhibitors were purified to homogeneity and subjected to MALDI-TOF-TOF-MS/MS analysis and N-terminal sequencing. Both ragi trypsin inhibitors (RTIs) showed the same N-terminal sequence and considerable sequence similarity to RBI, indicating the presence of a multigene protease inhibitor family in finger millet. To gain insight into the evolution of these genes, the upstream region of RBI was explored by Genome Walking. Interestingly, on sequencing, a genome walking product of ∼1 Kb showed presence of an N-terminal RBI specific primer sequence twice but in opposite directions and leaving an intervening region of ∼0.9 Kb. The intervening region was presumed to represent an E. coracana bidirectional promoter (EcBDP), intuitively having a divergent RBI-RTI gene pair at two sides. For assaying the bidirectionality of promoter activity, a dual reporter GUS-GFP vector construct was made for plant expression containing the reporter genes at two ends of EcBDP, which was used to transform Agrobacterium tumefaciens LBA 4404. Transient plant transformation by recombinant Agrobacterium cells was carried out in onion scale epidermal cells and finger millet seedling leaves. Simultaneous expression of GUS and GFP under EcBDP established it as a potent natural bidirectional promoter from monocot origin, thereby potentially having vast application in cereal gene manipulations. In addition, inducibility of the EcBDP by either abscisic acid or cold treatment, as determined by transient transformation in onion, would substantiate more precise control of gene expression to mitigate the effects of adverse environmental conditions
SMAR1 binds to T(C/G) repeatvand inhibits tumor progression by regulating miR-371-373 cluster
Get PDFChromatin architecture and dynamics are regulated by various histone and non-histone proteins. The
matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIPsequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse
biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the
availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional
repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-37
Role of bioactive components of ginger andgarlic in the reduction of Vibrio cholera induced infection in model systems
No full textCholera is an acute secretory diarrhoea caused by the Gram-negative bacterium Vibrio cholerae (Sack, Sack et al. 2004). In its severe form, cholera gravis, the clinical
disease is characterized by the passage of voluminous stools of rice water character that rapidly lead to dehydration. Hypovolemic shock, acidosis, and death can ensue in adults, as well as in children, if prompt and appropriate treatment is not initiated. Two
distinctive epidemiologic features of cholera are its tendency to appear in explosive outbreaks, often starting in several distinct foci simultaneously, and its propensity to cause true pandemics that progressively affect many countries in multiple continents
over the course of many year
Binding of the putative anticancer agent chelerythrine to double stranded poly(A): Calorimetry and spectral characterization studies
No full textThe benzophenanthridine plant alkaloid chelerythrine was recently reported to bind to single stranded
polyriboadenylic acid [ss poly(A)] through an entropy driven process with remarkably high binding affinity
(�107 M�1) (Basu and Suresh Kumar, 2015). Considering the anticancer effects of chelerythrine along with
its potential to be developed as an RNA targeted drug due to its high affinity to ss poly(A), here we studied
the binding of chelerythrine to double stranded (ds) poly(A). The binding was characterized thermodynamically
by enthalpy and entropy changes, and enthalpy-entropy compensation behaviour. The binding thermally stabilized the structure. The binding led to hypochromic and bathochromic effects in the visible absorption spectrum of chelerythrine and enhanced its fluorescence intensity. An intercalative binding mode was deduced from fluorescence quenching, anisotropy, and viscometric studies. The binding of chelerythrine to the ds poly(A) was significantly weaker than its binding to ss poly(A). The results may lead to designing RNA targeted therapeutics and a useful agent in gene regulation in eukaryotic cells
Dual hit’ metabolic modulator LDCA selectively kills cancer cells by efficient competitive inhibition of LDH-A†
No full textHerein, we synthesize and elucidate the potential of a novel ‘dual
hit’ molecule, LDCA, to constitutively block lactate dehydrogenase
isoform-A (LDH-A) to selectively subvert apoptosis and rigorously
attenuate breast tumor progression in amouse model, comprehensively
delineating the therapeutic prospectus of LDCA in the field of cancer
metabolic
Biophysical studies on the interaction of natural alkaloids and analogs with heme proteins
No full textAlkaloids represent an extensive group of secondary metabolites of plants with wide distribution in nature, diverse structures and with remarkable biological activities. Alkaloids and similar compounds with fused aromatic rings have the potential to form molecular
complexes with nucleic acids and proteins, and these are thought to be the cause of their therapeutic activity. Isoquinoline alkaloids such as berberine, palmatine, coralyne and sanguinarine (iminium and alkanolamine forms) have been established to have diverse biological and pharmacological activities and prospects to be developed as potential drugs for various therapies. Studies on the binding of drugs to proteins are of great importance in biological, biomedical and pharmaceutical sciences. Binding to proteins, particularly the transport proteins, is an
important element in determining the absorption, transport, distribution, cellular uptake, and activity of drugs in the circulatory systems. Binding parameters are indeed fundamental factors in determining the overall pharmacological activity of a drug. The concentration of
hemoglobin (Hb) in the blood stream (330 mg/mL) is much higher than those of the serum albumins (40 mg/mL). Even though Hb is not in the plasma, but will be available under
certain diseased conditions, it is important to study the interaction of potential drug candidates with Hb. An analysis of the complexation to Hb will provide informationon the pharmacological action and bioavailability of the drugs in the body. Furthermore, an
understanding of the details of the pharmaceutical interactions with heme proteins, hemoglobin ad myoglobin can also suggest new information on drug therapy and design as the second step in rational drug design.This thesis attempts to reveal the biophysical aspects of interaction of these protoberberine and benzophenanthridine alkaloids (isoquinolines) with heme proteins, using spectrophotometric, spectrofluorimetric and spectropolarimetric studies, and the thermodynamics of the interaction using isothermal titration calorimetry and differential scanning calorimetry tools. This thesis also aims to advance the knowledge by determining
the mode of binding to the proteins, correlating structural and energetic aspects on the interaction of these molecules with heme proteins for designing new natural product based
therapeutic molecules. Spectrophotometric studies showed that the interaction of these isoquinoline alkaloids with
heme proteins resulted in a hypochromic effect in the protein spectra and spectrofluorimetric studies revealed a significant quenching of the fluorescence of both proteins and the alkaloids. Föster resonance energy transfer (FRET) study showed that energy transfer from protein tryptophan residues (β-Trp36 of Hb) to the all alkaloids occurred with great possibility with the distances between the alkaloids and Trp of 2-8 nm in all cases. Circular dichroism, three dimensional fluorescence and synchronous fluorescence data suggested that binding of these alkaloids changed the conformation of the proteins, causing a reduction in
its -helical content. All the alkaloids were found to bind both heme proteins with moderate to high affinity with a binding constants ranging from 104-106 M-1. Overall the study suggested a higher binding affinity of sanguinarine iminium form to hemoglobin and coralyne to myoglobin.
Calorimetric studies were used to characterize the thermodynamics of the complexation.Enthalpy and entropy contributions at different temperatures were determined for all complexation. A unique phenomenon of enthalpy-entropy compensation was seen in some systems. The heat capacity changes derived from the temperature dependence of enthalpy
gave negative values in each system suggesting that the binding is specific and accompanied by the burial of non-polar surface area of proteins and also confirming the involvement of different interacting forces in the complexation. The results of structural and thermodynamic studies on the binding of these alkaloids to heme proteins provide important data in assessing the toxicity and risk factors in using these alkaloids as drugs and their distribution at the physiological sites. Furthermore, these results may help in the screening and design of appropriate isoquinoline based scaffolds that could be useful for developing as therapeutic agents
Role in proinflammatory response of YghJ, a secreted metalloproteasefrom neonatal septicemic Escherichia coli
No full textNeonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a sys-temic inflammatory response with production and release of a wide range of inflammatory mediators.The increased serum levels of cytokines were found to correlate with the severity and mortality in courseof sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflamma-tory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatalsepticemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant bysuccessive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showedhomology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ wasfound to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptidesubstrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7–8 and at temperatures37–40◦C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJstimulated the secretion of cytokines IL-1�, IL-1� and TNF-� in murine macrophages (RAW 264.7) andIL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates(54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) thanthe fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggeringproinflammatory response in NSEC
Elucidation of the molecular mechanisms of Vibrio cholerae virulence and biofilm gene induction following host cell contact.
No full textVibrio cholerae, etiological agent of the disease cholera, is known to form biofilms for persistence in
the environment. Previously it has been shown that host cell contact can induce virulence in adhered
bacteria. The effect of host cell contact on biofilm formation was however not known. It is
demonstrated here that about 7 fold upregulation of the biofilm regulatory gene vpsT was observed
within 30 minutes of adherence of V. cholerae to the intestinal cell line INT 407 and a massive
induction of about 700 fold was observed in rabbit ileal loops. The upregulation was observed in both
the classical and El Tor biotype strains of serogroup O1 that is most frequently associated with
epidemic cholera. Though a number of regulators are known to regulate vpsT in vitro, vpsT
upregulation was primarily dependent on the virulence master regulator AphA in adhered bacteria
with VpsR playing a secondary role. Quorum sensing regulator LuxO, secondary molecules c-di-
GMP or negative regulator HNS were found to have no role in vpsT upregulation. Though AphA is a
virulence master regulator, the induction of biofilms upon host contact appears to be independent of
virulence induction and a distinct temporal speciation exists between the two events. It was observed
that during infection, biofilm genes are upregulated almost immediately after host cell contact and
microscopic observation indicated that biofilm formation is initiated within 30 mins after adherence of
V. cholerae to intestinal cells. Of possible clinical relevance was the observation that V. cholerae in
the INT 407 associated biofilms was significantly more resistant to antibiotics than unadhered
planktonic cells which could lead to a number of complications during treatment of cholera. The
induction of vpsT was observed in vivo in rabbit ileal loops as well and it was found to be independant
of the strain or biotype of V. cholerae. Overall this work is the first direct evidence that V. cholerae
forms antibiotic resistant biofilm within the host in an entirely host contact dependent manner
Studies On The Role Of Cancer Stem Cells in Head And Neck Squamous Cell Carcinoma
No full textAlmost all epithelial tumors contain cancer stem-like cells, which possesses a unique property of self-renewal and differentiation. In oral cancer, several biomarkers including cell surface molecules have been exploited for the identification of this highly tumorigeneic population. Implicit is the role of CD44 in defining CSCs but CD24 is not well-explored in Oral Squamous Cell Carcinoma (OSCC). Here we show that CD44highCD24low cells isolated from the oral cancer cell lines, not only express stem cell related genes but also exhibit Epithelial-to-Mesenchymal transition (EMT) characteristics. This CD44highCD24low population gives rise to all other cell types upon differentiation. Typical Cancer Stem Cell (CSC) phenotypes like increased colony formation, sphere forming ability, migration and invasion were also confirmed in CD44highCD24low cells. These cells also showed enhanced resistance to apoptosis inducing stimuli owing to their slow-cycling state and high expression of drug transporters. Comparison with CD44highCD24high population revealed significantly enhanced stem-like characteristics of CD24low cells. To validate our findings in-vivo, we determined the relative expression of CD44 and CD24 in clinical samples of OSCC patients. CD44 expression was consistently high whereas CD24 showed significantly lower expression in tumor tissues. Moreover, whole transcriptome profiling reveals the gene-specific signatures of CSCs in OSCC. We conclude that CD44highCD24low represents cancer stem-like cells in Oral Squamous Cell Carcinoma. Deregulation of microRNAs (miRNAs) has been implicated in tumor development, but whether or how it regulates stemness in OSCC remains unclear. Here we report that miR-146a is significantly up-regulated in CSCs derived from oral cancer cell lines and primary tumors. Moreover, ectopic expression of miR-146a resulted in enhanced expression of CSC markers and self-renewing potential. Interestingly, miR-146a was found to induce CSC characteristics by stabilizing β-catenin that coincides with the loss of E-cadherin and CD24. Thus miR-146a is also linked with epithelial-mesenchymal transition (EMT) in addition to activated wnt signaling. Further, we identified and validated CD24 as a novel functional target of miR-146a and found that CD24 over-expression rescued miR-146a driven CSC properties. Detailed mechanistic investigations revealed an inverse relation between CD24 and β-catenin which is regulated by AKT activity. We hereby show that CD24 inhibits AKT leading to β-xx. catenin degradation. Our data also confirmed a positive feedback loop in β-catenin mediated trans-activation of miR-146a that possibly contributes to stem cell homeostasis. Taken together, our study suggests that miR-146a plays a critical role in regulating stemness properties in OSCC through CD24-AKT-β-catenin axis. Another means of epigenetic regulation that actively participate in cellular re-programming of cancer stem cells is bivalent chromatin modification. In this context, role of PRC2 (Polycomb Repressor Complex 2) has been found to be evident in mediating H3K27me3 modification, with effect on transcriptional repression of the developmental regulators. We have shown that EED, a component of PRC2, is moderately up-regulated in CSCs and when, transiently over-expressed in bulk cells, induces CSC markers. Based on our observations, we hypothesize that PRC2 promotes re-programming via Oct4highSox2low molecular signature, which probably defines the intermediate committed cells (progenitors) in OSCC. Here we have shown activation of Notch pathway to play an important role in mediating CSC-like phenotype, which is EZH2 dependent. Notch activation by PRC2 is probably the underlying mechanism of induced EMT and stemness in OSCC cells. Overall, the epigenetic means of CSC regulation emerge as a significant mechanism of modulating CSC plasticity. These results may provide insights into the CSC-based therapeutic intervention in oral cancers in general
Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway
Get PDFCytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of
different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different
apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of
annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect
on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It
has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therap