Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes

Background aimsHeart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation.MethodsWe treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)–polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation.ResultsWe observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium.ConclusionsThese studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.     

Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytes

We investigated the mechanisms whereby omega‐3 and ‐6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca²⁺]_i and reactive oxygen species (ROS). We applied the C‐22, 20, and 18 carbon omega‐3 (DHA, EPA, ALA) and omega‐6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA‐binding dye, propidium iodide. When applied at 20 µM concentration, omega‐3 fatty acids killed thymocytes over a period of 1 h with a potency of DHA > EPA > ALA. The omega‐6 PUFAs were more potent. The C18 omega‐6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca²⁺]_i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca²⁺]_i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca²⁺]_i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca²⁺]_i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca²⁺]_i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Substrate channeling in mammalian base excision repair pathways: passing the baton

The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or "hand-off" of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5'-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.     

Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.     

Energetics of gating at the apo–acetylcholine receptor transmitter binding site

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R↔R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates ∼50 Å to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the α (Y93, G147, W149, G153, Y190, C192, and Y198), ε (W55 and P121), or δ (W57) subunit. Gating energy changes were largest for the tryptophan residues. The αW149 energy changes were coupled with those of the other aromatic amino acids. Mutating the aromatic residues to Phe reduces the R/R* equilibrium dissociation constant ratio, with αY190 and αW149 being the most sensitive positions. Most of the mutations eliminated long-lived spontaneous openings. The results provide a foundation for understanding how ligands trigger protein conformational change.     

Excited state intramolecular proton transfer in 3-hydroxy flavone and 5-hydroxy flavone: A DFT based comparative study

Potential energy (PE) curves for the intramolecular proton transfer in the ground (GSIPT) and excited (ESIPT) states of 3-hydroxy-flavone (3HF) and 5-hydroxy-flavone (5HF) were studied using DFT/B3LYP (6-31G (d,p)) and TD-DFT/B3LYP (6-31G (d,p)) level of theory respectively. Our calculations suggest the non-viability of ground state intramolecular proton transfer for both the compounds. Calculated PE curves of 3HF for the ground and excited singlet states proton transfer process explain its four state laser diagram. Excited states PE calculations support the ESIPT process to both 5HF and 3HF. The difference in ESIPT emission process of 3HF and 5HF have been explained in terms of HOMO and LUMO electron distribution of the enol and keto tautomer of these two compounds.     

Effect of Bromocryptine on hormones and milk secretion in Murrah buffaloes ( Bubalus bubalis)

An experiment was carried out on 10 advance pregnant Murrah buffaloes to determine the role of hormones in milk secretion around parturition. Experimental animals were administered with a single injection of bromocryptine, @ 100 μg/kg BW, for 5 days before expected calving, whereas control group buffaloes were injected with placebo injections. Blood samples collected before parturition (-5,-4,-3,-2,-1 days), on day of parturition (day-0) and on day 1, 2, 3, 4, 5, 10 and 15 post partum were analyzed for growth hormone (GH), insulin like growth factor-I (IGF-I) and prolactin (PRL) by radioimmunassay methods. Milk samples were collected daily for 5 days and on day 10 and 15 after parturition. Milk fat, protein, lactose, citric acid, non-esterified fatty acids (NEFAs) and somatic cell counts (SCCs) were determined in milk samples. Bromocryptine treatment significantly (P < 0.01) decreased pre partum PRL and increased GH levels (P < 0.01) on day of parturition in experimental buffaloes without influencing plasma IGF-I level. Milk yield was significantly lower (P < 0.01) in experimental than in control group. Further, effect of bromocryptine on milk yield was only for a week. Milk yield increased (P < 0.01) gradually and was similar to control group on day 15 post partum. Bromocryptine treatment significantly increased milk SCC (P < 0.01) and protein content (P < 0.01) but there was no effect of treatment on fat, lactose, citric acid, glucose, milk and plasma NEFA concentration. It was concluded that prepartum suppression of PRL by bromocryptine impairs milk secretion temporarily in ensuing lactation. The significant rise in GH level before parturition and on day of parturition suggests a role of it in milk secretion of buffaloes.     

Anchor-Based Whole Genome Phylogeny (ABWGP): A Tool for Inferring Evolutionary Relationship among Closely Related Microorganims

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.