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1.  GBM's multifaceted landscape: highlighting regional and microenvironmental heterogeneity 
Neuro-Oncology  2014;16(9):1167-1175.
Gliomas are a heterogeneous group of tumors that show variable proliferative potential, invasiveness, aggressiveness, histological grading, and clinical behavior. In this review, we focus on glioblastoma multiforme (GBM), a grade IV glioma, which is the most common and malignant of primary adult brain tumors. Research over the past several decades has revealed the existence of extensive cellular, molecular, genetic, epigenetic, and metabolic heterogeneity among tumors of the same grade and even within individual tumors. Evaluation of different tumor types has shown that tumors with advanced grade and clinical aggressiveness also display enhanced molecular, cellular, and microenvironmental heterogeneity. From a therapeutic standpoint, this heterogeneity is a major clinical hurdle for devising effective therapeutic strategies for patients and challenges personalized medicine.
In this review, we will highlight key aspects of GBM heterogeneity, directing special attention to regional heterogeneity, hypoxia, genomic heterogeneity, tumor-specific metabolic reprogramming, neovascularization or angiogenesis, and stromal immune cells. We will further discuss the clinical implications of GBM heterogeneity in the context of therapy.
PMCID: PMC4136895  PMID: 24642524
angiogenesis; glioblastoma; hypoxia; metabolism
2.  Response of bacterioplankton to iron fertilization of the Southern Ocean, Antarctica 
Ocean iron fertilization is an approach to increase CO2 sequestration. The Indo-German iron fertilization experiment “LOHAFEX” was carried out in the Southern Ocean surrounding Antarctica in 2009 to monitor changes in bacterial community structure following iron fertilization-induced phytoplankton bloom of the seawater from different depths. 16S rRNA gene libraries were constructed using metagenomic DNA from seawater prior to and after iron fertilization and the clones were sequenced for identification of the major bacterial groups present and for phylogenetic analyses. A total of 4439 clones of 16S rRNA genes from ten 16S rRNA gene libraries were sequenced. More than 97.35% of the sequences represented four bacterial lineages i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Firmicutes and confirmed their role in scavenging of phytoplankton blooms induced following iron fertilization. The present study demonstrates the response of Firmicutes due to Iron fertilization which was not observed in previous southern ocean Iron fertilization studies. In addition, this study identifies three unique phylogenetic clusters LOHAFEX Cluster 1 (affiliated to Bacteroidetes), 2, and 3 (affiliated to Firmicutes) which were not detected in any of the earlier studies on iron fertilization. The relative abundance of these clusters in response to iron fertilization was different. The increase in abundance of LOHAFEX Cluster 2 and Papillibacter sp. another dominant Firmicutes may imply a role in phytoplankton degradation. Disappearance of LOHAFEX Cluster 3 and other bacterial genera after iron fertilization may imply conditions not conducive for their survival. It is hypothesized that heterotrophic bacterial abundance in the Southern Ocean would depend on their ability to utilize algal exudates, decaying algal biomass and other nutrients thus resulting in a dynamic bacterial succession of distinct genera.
PMCID: PMC4550105  PMID: 26379640
Southern Ocean; iron fertilization; metagenomic DNA; Major phylogenetic groups; unique bacterial clusters
3.  A preclinical study demonstrating the efficacy of nilotinib in inhibiting the growth of pediatric high-grade glioma 
Journal of Neuro-Oncology  2015;122(3):471-480.
Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRβ expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.
Electronic supplementary material
The online version of this article (doi:10.1007/s11060-015-1744-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4436849  PMID: 25732621
Pediatric glioma; Nilotinib; PDGFRα; AKT signaling; ERK1/2 signaling
4.  Withaferin A Alone and in Combination with Cisplatin Suppresses Growth and Metastasis of Ovarian Cancer by Targeting Putative Cancer Stem Cells 
PLoS ONE  2014;9(9):e107596.
Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed to chemo-resistance of cancer stem cells (CSCs). Herein we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera, when used alone or in combination with cisplatin (CIS) targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors generated by injecting human ovarian epithelial cancer cell line (A2780) with WFA and cisplatin (WFA) alone or in combination resulted in a 70 to 80% reduction in tumor growth and complete inhibition of metastasis to other organs compared to untreated controls. Histochemical and Western blot analysis of the tumors revealed that inclusion of WFA (2 mg/kg) resulted in a highly significant elimination of cells expressing CSC markers - CD44, CD24, CD34, CD117 and Oct4 and downregulation of Notch1, Hes1 and Hey1 genes. In contrast treatment of mice with CIS alone (6 mg/kg) had opposite effect on those cells. Increase in cells expressing CSC markers and Notch1 signaling pathway in tumors exposed to CIS may explain recurrence of cancer in patients treated with carboplatin and paclitaxel. Since, WFA alone or in combination with CIS eliminates putative CSCs, we conclude that WFA in combination with CIS may present more efficacious therapy for ovarian cancer.
PMCID: PMC4180068  PMID: 25264898
5.  Synthetic Triterpenoid Cyano Enone of Methyl Boswellate (CEMB) activates intrinsic, extrinsic, & endoplasmic reticulum stress cell death pathways in tumor cell lines 
Molecular cancer therapeutics  2011;10(9):1635-1643.
We explored the effect of a novel synthetic triterpenoid compound Cyano Enone of Methyl Boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD50) values in ten of ten tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways. By using small interfering RNAs (siRNAs), we show evidence that knock down of caspase 8, death receptor 4 (DR4), Apaf-1, and Bid impairs CEMB-induced cell death. Similar to other proapoptotic synthetic triterpenoid compounds, CEMB-induced apoptosis involved endoplasmic reticulum (ER) stress, as demonstrated by partial rescue of tumor cells by siRNA-mediated knock-down of expression of genes involve in the unfolded protein response such as Ire1, Perk, and ATF6. Altogether our results suggest that CEMB stimulates several apoptotic pathways in cancer cells, suggesting that this compound should be evaluated further as a potential agent for cancer therapy.
PMCID: PMC4174562  PMID: 21746806
Triterpenoid; Apoptosis; ER stress
6.  REST regulates oncogenic properties of glioblastoma stem cells 
Stem cells (Dayton, Ohio)  2012;30(3):405-414.
Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self-renewing, glioblastoma-derived stem-like cells (GSCs), the mechanisms that regulate self-renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor REST, suggesting heterogeneity across different GSC lines. Loss- and gain-of-function experiments indicated that REST maintains self-renewal of GSCs. High REST-expressing GSCs (HR-GSCs) produced tumors histopathologically distinct from those generated by low REST-expressing GSCs (LR-GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR-GSCs resulted in increased survival in GSC-transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR-GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR-GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self-renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM.
PMCID: PMC4039365  PMID: 22228704
REST; glioblastoma stem cells; neural stem cells; glioblastoma multiforme; invasion; self-renewal
7.  Interaction of Calcium-bound C-reactive Protein with Fibronectin Is Controlled by pH 
The Journal of biological chemistry  2004;279(50):10.1074/jbc.M409054200.
C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+-bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+-binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+-bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+-bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+-bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.
PMCID: PMC3819716  PMID: 15456743
8.  Role of the Property of C-Reactive Protein to Activate the Classical Pathway of Complement in Protecting Mice from Pneumococcal Infection1 
C-reactive protein (CRP) is not an acute-phase protein in mice, and therefore, mice are widely used to investigate the functions of human CRP. It has been shown that CRP protects mice from pneumococcal infection, and an active complement system is required for full protection. In this study, we assessed the contribution of CRP's ability of activating the classical pathway of complement in the protection of mice from lethal infection with virulent Streptococcus pneumoniae type 3. We used two CRP mutants, Y175A and K114A. The Y175A CRP does not bind C1q and does not activate complement in human serum. The K114A CRP binds C1q and activates complement more efficiently than wild-type CRP. Passively administered, both CRP mutants and the wild-type CRP protected mice from infection equally. Infected mice injected with wild-type or mutant CRP had reduced bacteremia, resulting in lower mortality and increased longevity compared with mice that did not receive CRP. Thus, the protection of mice was independent of CRP-mediated activation of the classical pathway of complement. To confirm that human CRP does not differentiate between human and mouse complement, we analyzed the binding of human CRP to mouse C1q. Surprisingly, CRP did not react with mouse C1q, although both mutant and wild-type CRP activated mouse C3, indicating species specificity of CRP-C1q interaction. We conclude that the mouse is an unfit animal for exploring CRP-mediated activation of the classical complement pathway, and that the characteristic of CRP to activate the classical complement pathway has no role in protecting mice from infection.
PMCID: PMC3818093  PMID: 16547275
9.  Human C-Reactive Protein Protects Mice from Streptococcus pneumoniae Infection without Binding to Pneumococcal C-Polysaccharide1 
Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcγ receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.
PMCID: PMC3818096  PMID: 17202380
10.  Auxarthronopsis, a new genus of Onygenales isolated from the vicinity of Bandhavgarh National Park, India 
IMA Fungus  2013;4(1):89-102.
An interesting onygenalean ascomycete was isolated from soil collected from a hollow tree near Bandhavgarh National Park situated in central India. The keratinophilic nature associated with a malbranchea-like asexual morph, appendaged mesh-like reticuloperidia, and subglobose to oblate, punctate ascospores, support the inclusion of this isolate in Onygenaceae. Further, the pale cream ascomata, punctate ascospores, and swollen septa in the peridial hyphae suggested that this was a new species of Auxarthron. However, phylogenetic study of LSU, SSU and ITS sequences, and presence of more than three swollen septa on the peridial appendages, do not support a placement within Auxarthron, and the new generic name Auxarthronopsis is introduced to accommodate this new fungus. The distinguishing features of this new taxon are the multiple (≥10) swollen septa on the appendages attached to its reticulate, loosely mesh-like peridium, the finely and regularly punctate ascospores, and the production of arthroconidial and aleurioconidial asexual forms. Sequence analysis of ITS1-5.8S-ITS2, SSU and LSU regions clearly separate this fungus from monophyletic Auxarthron and other taxa bearing some morphological similarity. Phylogenetically, Auxarthronopsis bandhavgarhensis gen. sp. nov. is closest to Amauroascus purpureus, A. volatilis-patellis, Nannizziopsis albicans, and Renispora flavissima, but differs morphologically.
PMCID: PMC3719210  PMID: 23898415
Auxarthron; Knuckle-joints; Molecular phylogeny; Multiseptate appendages; Onygenaceae
11.  A microRNA Link to Glioblastoma Heterogeneity 
Cancers  2012;4(3):846-872.
Glioblastomas (GBM) are one of the most malignant adult primary brain tumors. Through decades of research using various model systems and GBM patients, we have gained considerable insights into the mechanisms regulating GBM pathogenesis, but have mostly failed to significantly improve clinical outcome. For the most part GBM heterogeneity is responsible for this lack of progress. Here, we have discussed sources of cellular and microenvironmental heterogeneity in GBMs and their potential regulation through microRNA mediated mechanisms. We have focused on the role of individual microRNAs (miRNA) through their specific targets and miRNA mediated RNA-RNA interaction networks with the potential to influence various aspects of GBM heterogeneity including tumor neo-vascularization. We believe a better understanding of such mechanisms for regulation of GBM pathogenesis will be instrumental for future therapeutic options.
PMCID: PMC3712712  PMID: 24213470
glioma; glioblastoma; microRNA; angiogenesis; glioma stem cells; metabolism; Warburg effect
12.  Dynamic Status of REST in the Mouse ESC Pluripotency Network 
PLoS ONE  2012;7(8):e43659.
REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.
Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.
We show that Rest+/− and Rest−/− AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/− and Rest−/− AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest−/− AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency.
REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.
PMCID: PMC3429488  PMID: 22952733
13.  The connection between C-reactive protein and atherosclerosis 
Annals of Medicine  2008;40(2):110-120.
The connection between C-reactive protein (CRP) and atherosclerosis lies on three grounds. First, the concentration of CRP in the serum, which is measured by using highly sensitive (a.k.a. ‘hs’) techniques, correlates with the occurrence of cardiovascular disease. Second, although CRP binds only to Fcγ receptor-bearing cells and, in general, to apoptotic and damaged cells, almost every type of cultured mammalian cells has been shown to respond to CRP treatment. Many of these responses indicate proatherogenic functions of CRP but are being reinvestigated using CRP preparations that are free of endotoxins, sodium azide, and biologically active peptides derived from the protein itself. Third, CRP binds to modified forms of low-density lipoprotein (LDL), and, when aggregated, CRP can bind to native LDL as well. Accordingly, CRP is seen with LDL and damaged cells at the atherosclerotic lesions and myocardial infarcts. In experimental rats, human CRP was found to increase the infarct size, an effect that could be abrogated by blocking CRP-mediated complement activation. In the Apob100/100Ldlr -/- murine model of atherosclerosis, human CRP was shown to be atheroprotective, and the importance of CRP-LDL interactions in this protection was noted. Despite all this, at the end, the question whether CRP can protect humans from developing atherosclerosis remains unanswered.
PMCID: PMC3364506  PMID: 18293141
Atherosclerosis; cholesterol; C-reactive protein; foam cell; low-density lipoprotein; phosphoethanolamine; myocardial infarction
14.  Correction: Radiogenomic Mapping of Edema/Cellular Invasion MRI-Phenotypes in Glioblastoma Multiforme 
PLoS ONE  2012;7(2):10.1371/annotation/b5267cb3-6aa7-47fc-a648-47f30a7cff3e.
PMCID: PMC3270949
15.  Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate 
Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization.
The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml-1 at 72 h incubation. Enzyme production increased to 431 Uml-1 when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml-1 at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml-1 at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml-1 at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation.
The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml-1) in protease production compared to un-optimized conditions (65 Uml-1). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.
PMCID: PMC3337321  PMID: 22204659
16.  Radiogenomic Mapping of Edema/Cellular Invasion MRI-Phenotypes in Glioblastoma Multiforme 
PLoS ONE  2011;6(10):e25451.
Despite recent discoveries of new molecular targets and pathways, the search for an effective therapy for Glioblastoma Multiforme (GBM) continues. A newly emerged field, radiogenomics, links gene expression profiles with MRI phenotypes. MRI-FLAIR is a noninvasive diagnostic modality and was previously found to correlate with cellular invasion in GBM. Thus, our radiogenomic screen has the potential to reveal novel molecular determinants of invasion. Here, we present the first comprehensive radiogenomic analysis using quantitative MRI volumetrics and large-scale gene- and microRNA expression profiling in GBM.
Based on The Cancer Genome Atlas (TCGA), discovery and validation sets with gene, microRNA, and quantitative MR-imaging data were created. Top concordant genes and microRNAs correlated with high FLAIR volumes from both sets were further characterized by Kaplan Meier survival statistics, microRNA-gene correlation analyses, and GBM molecular subtype-specific distribution.
The top upregulated gene in both the discovery (4 fold) and validation (11 fold) sets was PERIOSTIN (POSTN). The top downregulated microRNA in both sets was miR-219, which is predicted to bind to POSTN. Kaplan Meier analysis demonstrated that above median expression of POSTN resulted in significantly decreased survival and shorter time to disease progression (P<0.001). High POSTN and low miR-219 expression were significantly associated with the mesenchymal GBM subtype (P<0.0001).
Here, we propose a novel diagnostic method to screen for molecular cancer subtypes and genomic correlates of cellular invasion. Our findings also have potential therapeutic significance since successful molecular inhibition of invasion will improve therapy and patient survival in GBM.
PMCID: PMC3187774  PMID: 21998659
17.  Atherosclerosis-related functions of C-reactive protein 
C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into monomers. Accordingly, the monomeric CRP found in atherosclerotic lesions may be a stationary, ligand-bound, by-product of a ligand-binding function of CRP. CRP binds to modified forms of low-density lipoprotein (LDL). The binding of CRP to oxidized LDL requires acidic pH conditions; the binding at physiological pH is controversial. The binding of CRP to enzymatically-modified LDL occurs at physiological pH; however, the binding is enhanced at acidic pH. Using enzymatically-modified LDL, CRP has been shown to prevent the formation of enzymatically-modified LDL-loaded macrophage foam cells. CRP is neither pro-atherogenic nor atheroprotective in ApoE−/− and ApoB100/100Ldlr −/− murine models of atherosclerosis, except in one study where CRP was found to be slightly atheroprotective in ApoB100/100Ldlr −/− mice. The reasons for the ineffectiveness of human CRP in murine models of atherosclerosis are not defined. It is possible that an inflammatory environment, such as those characterized by acidic pH, is needed for efficient interaction between CRP and atherogenic LDL during the development of atherosclerosis and to observe the possible atheroprotective function of CRP in animal models.
PMCID: PMC3125067  PMID: 20932269
Atherosclerosis; C-reactive protein; Enzymatically-modified low-density lipoprotein; Foam cells; Oxidized low-density lipoprotein
19.  Binding of the monomeric form of C-reactive protein to enzymatically-modified low-density lipoprotein: effects of phosphoethanolamine 
The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored.
We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL).
mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37°C, the binding of mCRP to E-LDL was enhanced <2-fold, while the binding of CRP to E-LDL was enhanced >10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37°C.
Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL.
PMCID: PMC2739981  PMID: 19545552
C-reactive protein; Monomeric C-reactive protein; Phosphoethanolamine; Pneumococcal C-polysaccharide; Enzymatically-modified low-density lipoprotein
20.  An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease 
Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min.
Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+) and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin). Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE) medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C.
An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.
PMCID: PMC2914678  PMID: 20646325
21.  DNA repair and recombination in higher plants: insights from comparative genomics of arabidopsis and rice 
BMC Genomics  2010;11:443.
The DNA repair and recombination (DRR) proteins protect organisms against genetic damage, caused by environmental agents and other genotoxic agents, by removal of DNA lesions or helping to abide them.
We identified genes potentially involved in DRR mechanisms in Arabidopsis and rice using similarity searches and conserved domain analysis against proteins known to be involved in DRR in human, yeast and E. coli. As expected, many of DRR genes are very similar to those found in other eukaryotes. Beside these eukaryotes specific genes, several prokaryotes specific genes were also found to be well conserved in plants. In Arabidopsis, several functionally important DRR gene duplications are present, which do not occur in rice. Among DRR proteins, we found that proteins belonging to the nucleotide excision repair pathway were relatively more conserved than proteins needed for the other DRR pathways. Sub-cellular localization studies of DRR gene suggests that these proteins are mostly reside in nucleus while gene drain in between nucleus and cell organelles were also found in some cases.
The similarities and dissimilarities in between plants and other organisms' DRR pathways are discussed. The observed differences broaden our knowledge about DRR in the plants world, and raises the potential question of whether differentiated functions have evolved in some cases. These results, altogether, provide a useful framework for further experimental studies in these organisms.
PMCID: PMC3091640  PMID: 20646326
22.  Chemical modifications of natural triterpenes - glycyrrhetinic and boswellic acids: evaluation of their biological activity 
Tetrahedron  2008;64(51):11541-11548.
Synthetic analogues of naturally occurring triterpenoids; glycyrrhetinic acid, arjunolic acid and boswellic acids, by modification of A-ring with a cyano- and enone- functionalities, have been reported. A novel method of synthesis of α-cyanoenones from isoxazoles is reported. Bio-assays using primary mouse macrophages and tumor cell lines indicate potent anti-inflammatory and cytotoxic activities associated with cyanoenones of boswellic acid and glycyrrhetinic acid.
PMCID: PMC2900779  PMID: 20622928
23.  REST/NRSF Maintains Self-Renewal and Pluripotency of Embryonic Stem Cells 
Nature  2008;453(7192):223-227.
The neuronal repressor REST/NRSF is expressed at high levels in mouse embryonic stem (mES) cells1, but its role in these cells has not been understood. Here we show that REST maintains self-renewal and pluripotency in mES cells through suppression of microRNA (miR)-21. We found that, as with known self-renewal markers, REST expression is much higher in self-renewing mES cells than in differentiating mES (mEB) cells. The heterozygous deletion of REST (REST+/−) and its siRNA-mediated knockdown in mES cells causes a loss of self-renewal, even when these cells are grown under self-renewal conditions, and leads to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mEB cells. In addition, REST+/− mES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4, Nanog, Sox2, and c-Myc, and exogenously added REST in mEB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We further show that in mES cells, REST is bound to the gene chromatin of a set of miRs that potentially target self-renewal genes. Whereas mES cells and mEB cells containing exogenously added REST express lower levels of these miRs, mEB cells, REST+/− mES cells, and siREST-treated mES cells express higher levels of these miRs. At least one of these REST-regulated miRs, miR-21, specifically suppresses the self-renewal of mES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2, and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mES cells.
PMCID: PMC2830094  PMID: 18362916
24.  Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases 
PLoS ONE  2009;4(8):e6851.
New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 µM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast (∼30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.
PMCID: PMC2731169  PMID: 19714254
25.  Phosphoethanolamine-complexed C-reactive protein: A pharmacological-like macromolecule that binds to native low-density lipoprotein in human serum 
C-reactive protein (CRP) is an acute phase plasma protein. An important binding specificity of CRP is for the modified forms of low-density lipoprotein (LDL) in which the phosphocholine-binding sites of CRP participate. CRP, however, does not bind to native LDL.
We investigated the interaction of CRP with native LDL using sucrose density gradient ultracentrifugation.
We found that the blocking of the phosphocholine-binding sites of CRP with phosphoethanolamine (PEt) converted CRP into a potent molecule for binding to native LDL. In the presence of PEt, CRP acquired the ability to bind to fluid-phase purified native LDL. Because purified native LDL may undergo subtle modifications, we also used whole human serum as the source of native LDL. In the presence of PEt, CRP bound to native LDL in serum also. The effect of PEt on CRP was selective for LDL because PEt-complexed CRP did not bind to high-density lipoprotein in the serum.
The pharmacologic intervention of endogenous CRP by PEt-based compounds, or the use of exogenously prepared CRP-PEt complexes, may turn out to be an effective approach to capture native LDL cholesterol in vivo to prevent the development of atherosclerosis.
PMCID: PMC2491905  PMID: 18486609
C-reactive protein; low-density lipoprotein; cholesterol; phosphocholine; phosphoethanolamine

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