Biomedical subjects

Sanghamitra Bandyopadhyay

Publications and source records attributed to Sanghamitra Bandyopadhyay.

9 recordsLinked to original sources

PubMed2006

Attenuation of osteoclastogenesis and osteoclast function by apigenin.

The physiological effects of the flavone, apigenin on bone cells were studied. We first show that apigenin inhibits tumor necrosis factor alpha (TNFalpha)- and interferon gamma (IFNgamma)-induced secretion of several osteoclastogenic cytokines from MC3T3-E1 mouse calvarial osteoblast cell line. Ligands of the TNF receptor family constitute the most potent osteoclastic cytokines. In MC3T3-E1 cells, apigenin dose-dependently (from 5 to 20 microM) inhibits TNFalpha-induced production of the osteoclastogenic cytokines, IL-6 (interleukin-6), RANTES (regulated upon activation, normal T cell-expressed and -secreted), monocyte chemoattractant protein-1 (MCP-1) and MCP-3. In addition, apigenin inhibits IFNgamma-stimulated secretion of monokines, CXCL-9, and -10 in MC3T3-E1 cells. Next, we show that apigenin strongly inhibits differentiation of 3T3-L1 preadipocytes to adipocytes with attendant inhibition of adipocyte differentiation-induced IL-6, MCP-1, and leptin production. Inhibition of adipogenic differentiation by apigenin could be due to induction of osteogensis as it robustly upregulates mRNA levels of bone morphogenetic protein-6 (BMP-6). Finally, the presence of apigenin inhibited osteoclast differentiation from the RAW 264.7 cell line by reducing receptor activator of nuclear factor kappa ligand (RANKL)-induced expression of tartrate-resistant acid phosphatase (TRAP), RANK, and calcitonin receptor but not CCR1, resulting in the inhibition of multinucleated osteoclast formation. Similarly, apigenin inhibited expression of the osteoclast differentiation markers TRAP, RANK, and c-Fms in osteoclast precursor cells obtained from mouse bone marrow following treatment with RANKL and macrophage colony stimulating factor (MCSF). Furthermore, apigenin induced apoptosis of mature osteoclasts obtained from rabbit long bone and inhibited bone resorption. In all instances, a structurally related compound, flavone had no significant effect. These data suggest that apigenin has multiple effects on all three bone cells that could prevent bone loss in vivo.

Animals↗

PubMed2006

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA.

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.

5' Untranslated Regions↗

PubMed2006

Interleukin-1alpha stimulates non-amyloidogenic pathway by alpha-secretase (ADAM-10 and ADAM-17) cleavage of APP in human astrocytic cells involving p38 MAP kinase.

Interleukin-1alpha (IL-1alpha) stimulates a disintegrin and metalloproteinase, ADAM-17 synthesis, consistent with activation of the soluble fragment of Amyloid Precursor Protein, APP, (sAPPalpha) in human primary astrocytes. To characterize the mechanism by which IL-1alpha promotes the non-amyloidogenic pathway of APP metabolism, we used U373 MG astrocytoma cells. IL-1alpha significantly increased levels of ADAM-10 and ADAM-17 mRNA in 16 hr. Upregulation of ADAM-17 mRNA by IL-1alpha was more pronounced despite higher basal levels of ADAM-10 mRNA. This pattern was also observed at the protein level with the upregulation of alpha-secretase. RNA interference (RNAi) of ADAM-10 and ADAM-17 inhibited IL-1alpha-stimulated sAPPalpha release and the effect was more pronounced with ADAM-17 RNAi. Concomitantly, the level of sAPPalpha was significantly increased by IL-1alpha in 48 hr; however, IL-1alpha stimulated cell-associated APP levels maximally at 6 h but the induction declined at 48 hr. IL-1alpha treatment of cells for 48 h reduced both intracellular and secreted levels of amyloid-beta, Abeta-40, and Abeta-42 peptides. Multiple MAP kinases (MAPK), including MEK/ERK, p38 kinase, PI3 kinase (PI3K) but not JNK were involved in the regulation of IL-1alpha-stimulated alpha-secretase activity and sAPPalpha release. p38 MAPK seems to be the most proximal of these MAPKs, as it was the earliest to be activated by IL-1alpha and blocking this pathway attenuated activation of IL-1alpha-induced MEK and PI3K pathways. Our data show a complex mechanism of sAPPalpha regulation by IL-1alpha that involves ADAM-10, ADAM-17 and p38 MAPK upstream of MEK and PI3K.

ADAM Proteins↗

PubMed2006

RNA therapeutics directed to the non coding regions of APP mRNA, in vivo anti-amyloid efficacy of paroxetine, erythromycin, and N-acetyl cysteine.

Lead compounds directed to the 5' leader of the Amyloid Precursor Protein transcript (i.e., paroxetine (SSRI), N-acetyl cysteine (antioxidant), and erythromycin (macrolide antibiotic)) were employed in a pilot study to evaluate their anti-amyloid efficacy in the TgCRND8 transgenic mouse model for Alzheimer's Disease (AD). The relative levels of Abeta peptide were reduced after exposure of mice to paroxetine (N=5), NAC (N=7), and erythromycin (N=7) relative to matched placebo counterparts. Paroxetine limited the levels of APP holoprotein and total Abeta peptide levels (measurements of Abeta were performed at two separate sites by quantitative western blotting and ELISA assay). The paroxetine data provided proof-of-concept for our strategy for further screening the APP 5'UTR target to identify novel drugs that exhibit anti-amyloid efficacy in vivo. Erythromycin and azithromycin were macrolide antibiotics that markedly changed the cleavage of the APP C-Terminal Fragment (CTF) in SH-SY5Y cells. Erythromycin provided orally to TgCRND8 mice consistently (100%) reduced brain Abeta(1-42) levels. These data demonstrated a highly statistically significant anti-amyloid trend for paroxetine, NAC and erythromycin. The potential for conducting further studies with these compounds using larger cohorts of TgCRND8 mice is discussed, particularly since erythromycin has recently been exposed to mice for a further 6 months (N=6). It will be possible to employ the chemical structures of paroxetine and erythromycin as starting points for drug design and development for AD therapeutics.

5' Untranslated Regions↗

PubMed2005

Functional expression of beta-chemokine receptors in osteoblasts: role of regulated upon activation, normal T cell expressed and secreted (RANTES) in osteoblasts and regulation of its secretion by osteoblasts and osteoclasts.

The expression and functions of receptors for the beta-chemokine, regulated upon activation, normal T cell expressed, and secreted (RANTES)/CCL5, were investigated in osteoblasts. Both primary osteoblasts and the MC3T3-E1 osteoblast cell line express the RANTES receptors, CCR1, 3, 4, and 5 (by RT-PCR), which encode functional receptors in osteoblasts as shown by [125I]-RANTES binding followed by Scatchard analysis. Expression of all four RANTES receptor mRNAs in osteoblast is in contrast to the reports of expression of CCR1 being the only RANTES receptor expressed by osteoclasts. Exogenous RANTES elicits chemotaxis of osteoblasts and promotes cell survival via phosphatidylinositol 3-kinase with attendant phosphorylation of Akt. Osteoclastic RANTES, obtained from the conditioned medium of receptor activator of nuclear factor-kappa B ligand-differentiated RAW264.7 cells also induces chemotaxis of MC3T3-E1 cells. Incubating the conditioned medium with an anti-RANTES neutralizing antibody attenuated this effect. RANTES secretion from osteoblast is inhibited by differentiation promoting hormones, e.g. 1,25 (OH)2D3 and dexamethasone, whereas macrophage inflammatory protein-1 alpha (but not macrophage inflammatory protein-1 beta) and elevated calcium induce it. Elevated calcium also stimulated RANTES secretion by osteoclasts. Therefore, RANTES is an osteoblast chemoattractant and a survival-promoting molecule whose regulation in osteoblast is varied. Furthermore, RANTES secreted from osteoclasts induces osteoblast chemotaxis. Therefore, expression of RANTES and its receptors in both osteoblasts and osteoclasts could enable this chemokine to act in autocrine/paracrine modes.

Animals↗

PubMed2005

Active site driven ligand design: an evolutionary approach.

An evolutionary approach for designing a ligand molecule that can bind to the active site of a target protein is described in this article. An earlier attempt in this regard assumed a fixed tree structure of the ligand on both sides of the pharmacophore, and used a genetic algorithm for optimizing the van der Waals energy. However, it is evident that knowledge about the size of the tree is difficult to obtain an a priori. Moreover, it will also change from one active site to another. This limitation is overcome in the present article by using variable string length genetic algorithm (VGA) for evolving an appropriate arrangement of the basic functional units of the molecule to be designed, whose size may now vary. The crossover and mutation operators are appropriately redesigned in order to tackle the concept of variable length chromosomes. Once the geometry of the molecule is obtained, the possible three-dimensional structure and its docking energy is determined. Results are demonstrated for five different target proteins both numerically and pictorially. It is found that not only does the molecule designed using variable length representation, in general, have lower energy values, the docking energies are also lower, as compared to the molecule evolved using fixed size representation.

Binding Sites↗

PubMed2004

Expression of pituitary tumor transforming gene (PTTG) and its binding protein in human astrocytes and astrocytoma cells: function and regulation of PTTG in U87 astrocytoma cells.

Human securin, pituitary tumor transforming gene (PTTG), is a protooncogene. Here we report expressions of PTTG and its interacting protein, PTTG-binding factor in human astrocytic cells. PTTG expression was higher in malignant cells than in primary astrocytes, whereas PTTG-binding factor was not. Using a xenotransplantable, glioma cell line (U87), we observed that knocking down PTTG mRNA by RNA silencing inhibited serum-induced proliferation by approximately 50%. Furthermore, in U87 cells PTTG expression was up-regulated by promalignant ligands epithelial growth factor (EGF) and TGFalpha, both at the protein and mRNA levels. PTTG induction by EGF receptor (EGFR) ligands could be blocked by the specific EGFR inhibitor, AG1478. Hepatocyte growth factor (HGF) also induced PTTG but to a lesser extent than EGF. Although EGF stimulates HGF secretion in U87 cells, the effect of EGF on PTTG mRNA expression is independent of HGF as neutralizing antibody against HGF failed to abolish EGF-induced up-regulation of PTTG mRNA. PTTG mRNA was unchanged by incubating U87 cells with the promalignant growth factor TGFbeta, apoptosis inducing TNFalpha and ligands for nuclear receptors, such as retinoic acid and retinoid X receptors and peroxisome proliferator-activated receptor-gamma, known for their growth-inhibitory and apoptosis-inducing effects on gliomas. In addition, 17beta-estradiol and Ca2+, known to activate PTTG expression, did not change PTTG mRNA levels in U87 cells. In summary, we show higher PTTG expression in astrocytoma than normal astrocytes and secondly, PTTG is involved in glioma cell growth. Finally, regulation of its expression has glioma-specific features and is selectively regulated by promalignant cytokines including EGFR ligands and HGF.

Apoptosis↗

PubMed2004

Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts.

The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca(2+)(o), a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca(2+)(o) did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca(2+)(o) did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca(2+)(o)-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca(2+)(o) is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca(2+)(o) stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.

Animals↗

PubMed2004

Multiobjective GAs, quantitative indices, and pattern classification.

The concept of multiobjective optimization (MOO) has been integrated with variable length chromosomes for the development of a nonparametric genetic classifier which can overcome the problems, like overfitting/overlearning and ignoring smaller classes, as faced by single objective classifiers. The classifier can efficiently approximate any kind of linear and/or nonlinear class boundaries of a data set using an appropriate number of hyperplanes. While designing the classifier the aim is to simultaneously minimize the number of misclassified training points and the number of hyperplanes, and to maximize the product of class wise recognition scores. The concepts of validation set (in addition to training and test sets) and validation functional are introduced in the multiobjective classifier for selecting a solution from a set of nondominated solutions provided by the MOO algorithm. This genetic classifier incorporates elitism and some domain specific constraints in the search process, and is called the CEMOGA-Classifier (constrained elitist multiobjective genetic algorithm based classifier). Two new quantitative indices, namely, the purity and minimal spacing, are developed for evaluating the performance of different MOO techniques. These are used, along with classification accuracy, required number of hyperplanes and the computation time, to compare the CEMOGA-Classifier with other related ones.

Algorithms↗