C-Phycocyanin,a selective cyclooxygenase-2 inhibitor,induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-Phycocyanin (C-PC) is one of the major biliproteins of Spirulina platensis, a blue green algae, with antioxidant and radical scavenging properties. It is also known to exhibit anti-inflammatory and anti-cancer properties. However, the mechanism of action of C-PC is not clearly understood. Previously, we have shown that C-PC selectively inhibits cyclooxygenase-2 (COX-2), an inducible isoform that is upregulated during inflammation and cancer. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of C-PC on LPS stimulated RAW 264.7 mouse macrophage cell line. These studies have shown a dose dependent reduction in the growth and multiplication of macrophage cell line by C-PC. This decrease in cell number appears to be mediated by C-PC induced apoptosis as evidenced by flow cytometric and confocal microscopic studies. Cells treated with 20 micro M C-PC showed typical nuclear condensation and 16.6% of cells in sub-G(o)/G(1) phase. These cells also showed DNA fragmentation in a dose dependent manner. The studies on poly(ADP ribose) polymerase (PARP) cleavage showed typical fragmentation pattern in C-PC treated cells. This C-PC induced apoptosis in RAW 264.7 cells appears to be mediated by the release of cytochrome c from mitochondria and independent of Bcl-2 expression. These effects of C-PC on RAW 264.7 cells may be due to reduced PGE(2) levels as a result of COX-2 inhibition.     

Megafaunal community structure of Andaman seamounts including the Back-arc Basin--a quantitative exploration from the Indian Ocean

Species rich benthic communities have been reported from some seamounts, predominantly from the Atlantic and Pacific Oceans, but the fauna and habitats on Indian Ocean seamounts are still poorly known. This study focuses on two seamounts, a submarine volcano (cratered seamount--CSM) and a non-volcano (SM2) in the Andaman Back-arc Basin (ABB), and the basin itself. The main purpose was to explore and generate regional biodiversity data from summit and flank (upper slope) of the Andaman seamounts for comparison with other seamounts worldwide. We also investigated how substratum types affect the megafaunal community structure along the ABB. Underwater video recordings from TeleVision guided Gripper (TVG) lowerings were used to describe the benthic community structure along the ABB and both seamounts. We found 13 varieties of substratum in the study area. The CSM has hard substratum, such as boulders and cobbles, whereas the SM2 was dominated by cobbles and fine sediment. The highest abundance of megabenthic communities was recorded on the flank of the CSM. Species richness and diversity were higher at the flank of the CSM than other are of ABB. Non-metric multi-dimensional scaling (nMDS) analysis of substratum types showed 50% similarity between the flanks of both seamounts, because both sites have a component of cobbles mixed with fine sediments in their substratum. Further, nMDS of faunal abundance revealed two groups, each restricted to one of the seamounts, suggesting faunal distinctness between them. The sessile fauna corals and poriferans showed a significant positive relation with cobbles and fine sediments substratum, while the mobile categories echinoderms and arthropods showed a significant positive relation with fine sediments only.     

Thioacetamide-Induced Fulminant Hepatic Failure Induces Cerebral Mitochondrial Dysfunction by Altering the Electron Transport Chain Complexes

Fulminant hepatic failure (FHF) is an acute form of hepatic encephalopathy resulting from severe inflammatory or necrotic liver damage without any previously established liver damage. This develops as a complication due to viral infections, and drug abuse. FHF also occurs in acute disorders like Reye’s syndrome. Although the exact mechanisms in the etiology of FHF are not understood, elevated levels of brain ammonia have been consistently reported. Such increased ammonia levels are suggested to alter neurotransmission signals and impair cerebral energy metabolism due to mitochondrial dysfunctions. In the present study we have examined the role of cerebral electron transport chain complexes, including complex I, II, III IV, and pyruvate dehydrogenase in the non-synaptic mitochondria isolated from the cortex of the thioacetamide-induced FHF rats. Further, we have examined if the structure of mitochondria is altered. The results of the current study demonstrated a decrease in the activity of the complex I by 31 and 48% at 18 and 24 h respectively after the thioacetamide injection. Similarly, the activity of electron transport chain complex III was inhibited by 35 and 52% respectively, at 18 and 24 h, respectively. The complex II and complex IV, on the other hand, revealed unaltered activity. Further the activity of pyruvate dehydrogenase at 18 and 24 h after the induction of FHF was inhibited by 29 and 43%, respectively. Our results also suggest mitochondrial swelling in FHF induced rats. The inhibition of the respiratory complexes III and I and pyruvate dehydrogenase might lead to the increased production of free radical resulting in oxidative stress and cerebral energy disturbances thereby leading to mitochondrial swelling and further contributing to the pathogenesis of FHF.     

Dietary Docosahexaenoic Acid Supplementation Modulates Hippocampal Development in the Pemt?/? Mouse

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt^−/− mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt^−/− mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt^−/− dams fed the control diet had 25–50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt^−/− phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt^−/− mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.     

Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-1-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.     

Effect of periplasmic nitrate reductase on diauxic lag of Paracoccus pantotrophus

Paracoccus pantotrophus expresses two nitrate reductases—membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap^? mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Micropropagation of Three Varieties of Caralluma adscendens via Nodal Explants

A procedure for in vitro propagation of pharmaceutically valuable varieties of Caralluma adscendens from nodal explant, is described. The highest shoot multiplication with 80% frequency was achieved within one month on Murashige and Skoog’s medium supplemented with 8.87 μM BA. Shoot multiplication occurred in subsequent subcultures in culture bottles on MS medium. Regenerated shoots were rooted on half strength MS medium supplemented with NAA (0.54 μM) in all the three varieties. The rooted plants were hardened for establishment in soil.     

Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth

Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplemention with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.     

Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas

Background

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.

Methodology/Principal Findings

In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.

Conclusions/Significance

In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.     

Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer cell growth by inhibiting ErbB-2 and ERK1

A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by performing in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore, the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal human fibroblasts suggesting that these compounds are tumor-specific.