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Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation
No full textCancer treatment using gold (I) complexes is becoming popular. In this study, a gold (I) N-heterocyclic
complex designated as complex 3 was synthesized, its cytotoxicity was examined, and its anti-melanoma activity wasevaluated in vitro and in vivo. Viability of cancer cells was determined by MTT assay upon treatment with various concentrations of a gold (I) N-heterocyclic carbene complex (complex 3) in a dose and time dependent manner. Mouse melanoma cells B16F10 were selected for further apoptotic studies, including flowcytometric analysis of annexin binding, cell cycle arrest, intracellular ROS generation and loss in the mitochondrial membrane potential. ELISA based assays were done for
caspase activities and western blots for determining the expression of various survival and apoptotic proteins.
Immunocytology was performed to visualize the translocation of p53 to the nucleus. B16F10 cells were inoculated
into mice and post tumor formation, complex 3 was administered. Immunohistology was performed to determine the
expressions of p53, p21, NF-κB (p65 and p50), MMP-9 and VEGF. Student’s t test was used for determining statistical
significance. The survival rate data were analyzed by Kaplan-Meier plots.Complex 3 markedly inhibited the growth of HCT 116, HepG2, and A549, and induced apoptosis in B16F10
cells with nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, activation of caspase 3 and caspase 9, PARP cleavage, downregulation of Bcl-2, upregulation of Bax, cytosolic cytochrome c elevation, ROS generation, and mitochondrial membrane potential loss indicating the involvement of an intrinsic mitochondrial death pathway. Further, upregulation of p53, p-p53 (ser 15) and p21 indicated the role of p53 in complex 3 mediated apoptosis. The complex reduced tumor size, and caused upregulation of p53 and p21 along with downregulation of NF-κB (p65 and p50), VEGF and MMP-9. These results suggest that it induced anti-melanoma effect in vitro and in vivo by modulating p53 and other apoptotic factors. The gold (I) N-heterocyclic carbene complex (C22H26N6AuO2PF6) designated as complex 3 induced
ROS and p53 dependent apoptosis in B16F10 cells involving the mitochondrial death pathway along with suppression of melanoma tumor growth by regulating the levels of pro and anti apoptotic factors (p53, p21, NF-κB, VEGF and MMP-9)
Proteomic profiling of γ-ECS overexpressed transgenic Nicotiana in response to drought stress
No full textThe contribution of Glutathione (GSH ) in drought stress tolerance is an established fact. However, the proteins
which are directly or indirectly related to the increased level of GSH in response to drought stress are yet to be
known. To explore this, here, transgenic tobacco plants (NtGp11) overexpressing gamma-glutamylcysteine synthetase
(γ-ECS) was tested for tolerance against drought stress. NtGp11 conferred tolerance to drought stress by increased
germination rate, water retention, water recovery, chlorophyll, and proline content compared with wild-type plants.
Semi-quantitative RT-PCR analysis revealed that the transcript levels of stress-responsive genes were higher in
NtGp11 compared with wild-type in response to drought stress. Two-dimensional gel electrophoresis (2-DE) coupled
with MALDI TOF-TOF MS/MS analysis has been used to identify 43 differentially expressed proteins in response to
drought in wild-type and NtGp11 plants. The results demonstrated the up-accumulation of 58.1% of proteins among
which 36%, 24%, and 20% of them were related to stress and defense, carbon metabolism and energy metabolism
categories, respectively. Taken together, our results demonstrated that GSH plays an important role in combating
drought stress in plants by inducing stress related genes and proteins like HSP70, chalcone synthase, glutathione
peroxidase, thioredoxin peroxidase, ACC oxidase, and heme oxygenase I
Biochemical Characterization of a Novel Heme-based Adenylate Cyclase from Leishmania major
No full textThrough the billion years of evolution of the earth’s biosphere, chemistry of the atmosphere changed from a primitive unbreathable mixture of gas to oxygen-rich air. According to the geologic time scale, in the Neoproterozoic-Cambrian period there was a major rise in atmospheric oxygen, and the start of initial evolution and subsequent ‘explosion’ of complex life. Since then the aerobic life, from tiny bacteria to civilized human, absolutely depends on the oxygen for survival. To harness the atmospheric oxygen in beneficial way, globins are evolved in the living system. Globins are a family of globular heme-containing protein that also shares billion years of evolutionary history. Globins occur in all three kingdoms of life and can be classified into single-domain globins and chimeric globins. The latter comprise the globin-coupled sensors (GCS) that is a chimeric protein, composed of oxygen-sensing/binding globin domain and functional/catalytic domain. Until our work, it was thought that GCSs are present only in bacteria and archaea, and absent in eukaryotes. We have discovered a unique GCS (HemAC-Lm) made of neuroglobin/cytoglobin like domain coupled with adenylate cyclase domain in unicellular eukaryotic organism Leishmania major. Hypoxia is a condition in which the tissue or a region of the body is deprived of adequate oxygen supply. Hypoxia induced cell death has been implicated in a number of pathophysiological conditions like heart disease, cancer, cerebrovascular disease, and chronic lung disease, which are responsible for two-thirds of all deaths annually worldwide. Although transcriptional activator hypoxia-inducible factor 1 (HIF-1) in mammals plays a key role in hypoxia mediated adaptive responses, the exact mechanism of hypoxia induced cell death is obscure till date. Second messenger 3’,5’-cyclic adenosine monophosphate (cAMP) is known to play an important role in a number of metabolic and developmental processes in all living organisms. However the role of cAMP signalling in hypoxia induced cell death is little bit contradictory. Due to the very unique domain architecture, fusion of heme-bound globin and adenylate cyclase domains, HemAC-Lm may hold the answer to actual role of cAMP signalling during hypoxia. Leishmaniasis remains one of the world's most devastating neglected tropical diseases, causing substantial mortality or permanent deformity to nearly 12 million affected individual worldwide. Despite of the prevalence of the disease there is no vaccine or prophylactic drug available to treat the disease. Current treatments for the leishmaniasis are unsatisfactory due to their route of administration, toxicity, expense and the widespread resistance to first-line antimonial drugs. Therefore we are in a desperate search for new drug targets and effective chemotherapeutic agents against leishmaniasis. cAMP signalling in Leishmania is thought to play an important role in its survival and infectivity and like several other diseases as Parkinson’s disease, schizophrenia, asthma, diabetes, this signalling pathway can also be used as potential antileishmanial drug targets. The exclusivity of HemAC-Lm among living being, provide the opportunity to investigate possible mechanisms of inhibition in the framework of novel drug target discovery. In this dissertation an effort is made to unravel the secrets of HemAC-Lm through the biochemical and functional characterization. The findings of our work are discussed in three consecutive chapters. Chapter 1 presents the cloning, expression, biochemical and structural characterization of HemAC-Lm in detail. Chapter 2 deals with physiological significance of the protein under normoxic condition by generating half knockout, conditional knockdown and overexpressing strains corresponding to the HemAC-Lm gene. Chapter 3 deals with essentiality of HemAC-Lm under hypoxic condition and how increase or decrease in its intracellular concentration leads to ROS mediated cell death within L. major
Matrix Metalloproteinase-1 (MMP-1) Promoter Polymorphisms are Well Linked with Lower Stomach Tumor Formation in Eastern Indian Population
Get PDFExpression of matrix metalloproteinase-1 (MMP-1), an interstitial collagenase, plays a major role in cellular invasion during development of gastric cancer, a leading cause of death worldwide. A single-nucleotide polymorphism (SNP) 21607 1G/2G site of the MMP-1 gene promoter has been reported to alter transcription level. While the importance’s of other SNPs in the MMP-1 promoter have not yet been studied in gastric cancer, our aim was to investigate MMP-1 gene promoter polymorphisms and gastric cancer susceptibility in eastern Indian population. A total of 145 gastric cancer patients and 145 healthy controls were genotyped for MMP-1 21607 1G/2G (rs1799750) by PCR-restriction fragment length polymorphism (RFLP), while MMP-1 2519 A/G (rs1144393), MMP-1 2422 T/A (rs475007), MMP-1 2340 T/C (rs514921) and MMP-1 2320 T/C (rs494379) were genotyped by DNA sequencing. A positive association was found with MMP-1 2422 T/A SNP that showed significant risk for regional lymph node metastasis (P = 0.021, Odd’s ratio (OR) = 3.044, Confidence intervals (CI) = 1.187– 7.807). In addition, we found a significant association with lower stomach tumor formation among gastric cancer patients for three adjacent polymorphisms near the transcriptional start sites of [MMP-1 2422 T/A (P = 0.043, OR = 2.182, CI = 1.03– 4.643), MMP-1 2340 T/C (P = 0.075, OR = 1.97, CI = 0.94–4.158) and MMP-1 2320 T/C (P = 0.034, OR = 2.224, CI = 1.064– 40731)]. MMP-1 level in patients’ serum was correlated with MMP-1 promoter haplotypes conferring these three SNPs to evaluate the functional importance of these polymorphisms in lower stomach tumor formation and significant correlation was observed. Furthermore, MMP-1 2519 A/G polymorphism displayed poor cellular differentiation (P = 0.024, OR = 3.8, CI = 1.69–8.56) attributing a higher risk of cancer progression.In conclusion, MMP-1 proximal promoter SNPs are associated
with the risk of lower stomach tumor formation and node metastasis in eastern Indian population
Diarrhoeal Health Risks Attributable to Water-Borne-Pathogens in Arsenic-Mitigated Drinking Water in West Bengal are Largely Independent of the Microbiological Quality of the Supplied Water
No full textThere is a growing discussion about the possibility of arsenic mitigation measures in Bengal and similar areas leading to undesirable substitution of water-borne-pathogen
attributable risks pathogens for risks attributable to arsenic, in part because of uncertainties in relative pathogen concentrations in supplied and end-use water. We try to resolve thisdiscussion, by assessing the relative contributions of water supply and end-user practices to water-borne-pathogen-attributable risks for arsenic mitigation options in a groundwater arsenic impacted area of West Bengal. Paired supplied arsenic-mitigated water and end-use drinking water samples from 102 households were collected and analyzed for arsenic and thermally tolerant coliforms [TTC], used as a proxy for microbiological water quality, We then estimated the DALYs related to key sequelae, diarrheal diseases and cancers, arising
from water-borne pathogens and arsenic respectively. We found [TTC] in end-use drinking water to depend only weakly on [TTC] in source-water. End-user practices far outweighed
the microbiological quality of supplied water in determining diarrheal disease burden. [TTC] in source water was calculated to contribute <1% of total diarrheal disease burden. No substantial demonstrable pathogen-for-arsenic risk substitution attributable to specific arsenic mitigation of supplied waters was observed, illustrating the benefits of arsenic mitigation measures in the area studied
Genomic And Proteomic Studies On Arsenic Toxicity
No full textbe positively correlated with their respective intracellular ROS level, which is consistent with
the significant variation found in ND3 gene among the exposed individuals. In addition, the extent of potentially damaging variants in arsenic-exposed individuals had significant positive correlation to the degree of G0/G1 cell cycle arrest.In the context of understanding the underlying molecular mechanisms that render a person susceptible to arsenic toxicity it was imperative to look for the differentially expressed proteins (a) among the arsenic exposed and unexposed individuals, and (b) among the
arsenic exposed individuals with and without skin lesion. To address this problem, plasma samples from 40 individuals expose to arsenic (20 symptomatic, 20 asymptomatic) and 20
unexposed individuals were taken as discovery cohort and subjected to iTRAQ followed by mass spectrometry analysis. A replicative cohort comprising of total 30 individuals (10
symptomatic, 10 asymptomatic and 10 unexposed) were also similarly subjected to iTRAQ based protein quantitation. A total of 103 proteins were identified to be differentially expressed among the three groups in both the discovery and replicative cohorts. The proteins thus identified were enriched in several biological processes like apoptosis, cellular adhesion,endothelial processes, immune responses, etc. defined by Gene Ontology terms and determined by online free software Genecodi(http://genecodis.cnb.csic.es/). Further, OMIM
analysis revealed that defects in genes like APOE and DSG1 lead to skin lesion. The “skin lesion phenotype” caused due to defects in DSG1 is very similar to the type of skin lesion caused by arsenicosis. To check whether genetic defect of this gene is actually related to arsenic induced skin lesion, the entire DSG1 gene along with its regulatory regions were screened in 25 symptomatic and 25 asymptomatic individuals. In this pilot study no significant change was observed in the DSG1 gene. Interestingly, APOE isoforms have been recently reported to be associated with cardiovascular disease in the arsenic exposed individuals of
Taiwan. When the APOE isoforms as well as the promoter SNPs were determined in our population of arsenic exposed individuals (100 symptomatics and 100 asymptomatics)
significant association was observed. Further, DSG1, DAPK3 and APOE, found to be differentially expressed by iTRAQ, were validated by Western Blot in individual plasma
samples
Role of Wnt5a-Fz5 Signaling In Innate Immunity
No full textInnate immunity provides the first line of defense against infection from various microorganisms. Among the different types of cells involved in innate immunity, macrophages play a very significant role. The presence of differentiation antigen such as CD14 on of macrophage surface is an important feature of macrophage mediated immune functions. How macrophages remain in such groomed condition in our system to counter infections remains an enigmatic question. The signaling pathways of homeostatic circuitry that keeps macrophages prepared to combat invasion remain unresolved. Although monocytic cells as well as macrophages harbor nuclear NFкB, it is unclear whether this nuclear NFκB engages in preparing macrophages for immune defense. I investigated (i) if a constitutive nuclear NFκB pool contributes to sustain macrophage mediated immune response and (ii) what cellular circuitry is responsible for such homeostasis. Because Wnt5a-Fz5 signaling is a primordial event during cell differentiation and has previously been shown to be involved in inflammation and phagocytosis, I examined the involvement of Wnt5a-Fz5 signaling in the constitutive presence of nuclear NFκB (p65) as well as maintenance of innate immune functions in macrophages. I demonstrated that a homeostatic Rac1 dependent Wnt5a-Fz5-NFκB (p65) circuit is at least partly responsible for keeping macrophages prepared for immune response. The autocrine/paracrine Wnt5a-Fz5-Rac1-p65 signaling cascade not only maintains basal levels of the immune defense modulating cytokines/interferons and CD14, but also supports macrophage survival. This homeostatic circuit accordingly drives the natural immune response of macrophages to E. coli/LPS and virus. The depiction of a functional cytokine/interferon response both in vitro and in vivo as an outcome of a homeostatic Wnt5a-p65 axis unfolds previously unidentified details of immune regulation in macrophages. Additionally, I demonstrated that Wnt5a signalling also contributes to monocytes survival and differentiation into macrophages
Expression of the major virulence genes of the bacterium Helicobacter pylori following adherence to human gastric cells
No full textHelicobacter pylori, a gram-negative bacterium, colonizes the human stomach in a majority of
the world’s population. The major virulence factors of H. pylori, CagA, VacA are thought to be
associated with chronic inflammation and disease and BabA is necessary for adherence of the
bacterium to host cells. These virulence factors have been extensively studied but the regulation
of expression of these virulence genes in H. pylori remains poorly understood. In this study, H.
pylori was allowed to adhere to a gastric epithelial cell line (AGS) and expression of the
virulence genes was examined in the adhered H. pylori by quantitative RT-PCR. Adherence of
H. pylori to the gastric epithelial cell line AGS, strongly induces expression of the major
virulence genes in H. pylori (cagA, vacA and babA). The induction was dependent on the iron
sensing transcription factor Fur which acts as a global regulator in H. pylori. The induction of
these virulence factors in the AGS cell-adhered H. pylori Δfur mutant strain was consistently
lower than in the adhered parent strain. However expression of the genes was similar between
the wild type and Δfur mutant strains in the un-adhered state, suggesting that Fur has a role in the
upregulation of cagA, vacA and babA expression specifically in host cell adhered H. pylori. The
Δfur deletion mutant in H. pylori was constructed by splicing by overlap extension (SOE) PCR
and allelic exchange method. Furthermore it has been demonstrated that, iron-cofactored Fur
functions as a transcriptional activator of cagA and vacA in host cell adhered H. pylori.
Consistent with these results, microscopic observations revealed that infection of AGS cells with
H. pylori Δfur mutant strain produced much less damage as compared to that produced by the
wild type H. pylori strain. Taken together, these results suggest that cagA, vacA and babA genes
are upregulated in H. pylori specifically by host cell contact and Fur has a role in the
upregulation
Structural characterization of Type III Secretion System related translocator and effector from Pseudomonas aeruginosa
No full textPseudomonas aeruginosa, a Gram-negative pathogen utilizes a specialized set of Type III
Secretion System (T3SS) translocator and effector proteins to establish virulence in the host
cell. An understanding of these pathogenic factors that play a key role in the establishment
and maintenance of bacterial pathogenicity are thus, of immense importance, and are likely
the interest of study of several research groups.
The T3SS encoding “translocator operon” of P. aeruginosa consists of a major translocator
protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. We
present a comprehensive study of PopB structure, its interaction with PcrH and their
associated pH-based structural and functional changes. The pH-dependent studies indicate
that PcrH not only provides structural support to the ordered molten globule PopB in
complex but also undergoes conformational change to assist PopB to pass through the needle
complex of T3SS and form pores in the host cell membrane. In addition, we report an abinitio
model of PopB docked to PcrH, which together with PCR-based deletion mutations and
SPR studies determine the terminal domains of PopB to be involved in hydrophobic
interaction with PcrH as well as maintain the oligomerisation of translocator complex.
Amongst all effector toxin proteins of T3SS present in P. aeruginosa, both ExoT and ExoS
are considered to be actual virulence determinants. Here, we report the first X-ray crystal
structure of the amino-terminal fragment of effector toxin ExoT, in complex with full-length
homodimeric chaperone SpcS at 2.1Å resolution. The full-length dimeric chaperone SpcS has
the conserved α-β-β-β-α-β-β-α fold of class I chaperones, the characteristic hydrophobic
patches for binding effector proteins and a conserved polar cavity at the dimeric interface.
The stable crystallized amino-terminal fragment of ExoT consists of a chaperone binding
domain and a membrane localization domain that wraps around the dimeric chaperone. Sitedirected
mutagenesis experiments and molecular dynamics study complement each other in
revealing Asn65, Phe67 and Trp88 as critical dimeric interfacial residues that can strongly
influence the effector-chaperone interactions.
This structural and functional T3SS study can thus serve to be promising drug candidates to
combat host cell infections
Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa
No full textPseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein–chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone
PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a “disorder-to-order” transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism