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Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca 2 + sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin–myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca 2 + -stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca 2 + transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca 2 + . These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca 2 + -stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca 2 + , while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca 2 + -stimulated ATPase activity, which subsequently suppresses myocardial contractile function through a decrease of myofilament Ca 2 + sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: Interactions between the proteasome inhibitor, bortezomib, and the sphingosine kinase (SPK1) inhibitor, SKI, were examined in BCR/ABL human leukemia cells. Coexposure of K562 or chronic myeloid leukemia (CML) cells from patients to subtoxic concentrations of SKI (10μM) and bortezomib (100nM) resulted in a synergistic increase in caspase-3 cleavage and apoptosis. These events were associated with the downregulation of BCR–ABL and Mcl-1, and a marked reduction in SPK1 expression. In imatinib mesylate-resistant K562 cells that displayed decreased BCR–ABL expression, bortezomib/SKI treatment markedly increased apoptosis and inhibited colony-formation in association with the downregulation of Mcl-1. Finally, the bortezomib/SKI regimen also potently induced the downregulation of BCR/ABL and Mcl-1 in human leukemia cells. Collectively, these findings suggest that combining SKI and bortezomib may represent a novel strategy in leukemia, including apoptosis-resistant BCR–ABL+ hematologic malignancies. [Copyright &y& Elsevier]

Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation. [ABSTRACT FROM AUTHOR]

Abstract: 2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a “slipstreaming” mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression. [Copyright &y& Elsevier]

The study was designed to elucidate roles of PKCepsilon, ERK1/2, STAT3 in regulations of endogenous H2S in rat hepatocyte apoptosis induced by LPS. The cultured rat hepatocytes were treated with vehicle or different dose of LPS respectively. In the separate experiments, hepatocytes were treated with CBS inhibitor AOAA,CSE inhibitor PAG or PKC epsilon, ERK1/2, STAT3 specific inhibitors SCP0213, A6355, S31-201,respectively or in a different combination manner. PKC epsilon, ERK1/2 and STAT3 phospharylations, PKCepsilon membranous translocation, STAT3 nuclear translocation were detected with Western blotting. Apoptosis rates were detected with flow cytometry. PKC epsilon, ERK1/2 and STAT3 phosphorylation expression in LPS-treated hepatocytes were significantly up-regulated, obviously in 30min compared with vehicle,furthermore membranous tranlocation of PKCepsilon and nuclear tranlocation of STAT3 were found. Compared with LPS, AOAA or PAG combined with LPS respectively or jointly could cause further more obvious up-regulation in phosphorylative levels of PKCepsilon, ERK1/2 or STAT3 protein expression. SCP0213, A6355 or S31-201 combined with LPS respectively could cause increased apoptosis rates in 4h and 12h compared with LPS alone. When hepatocytes were treated with SCP0213, A6355 or S31-201 combined with AOAA or PAG respectively, apoptosis rates increased in 12h but no significant changes in 4h compared with AOAA or PAG+LPS treatments.Compared with LPS,SCP0213+LPS could cause down-regulation in phosphorylative levels of ERK1/2 or STAT3 protein expressions, A6355+LPS could cause down-regulation in the phosphorylative level of STAT3 protein expression without change in PKCepsilon, S31–201+LPS could not cause any changes of phosphorylative level of protein expression in PKCepsilon or ERK1/2. In conclusion,PKCepsilon-ERK1/2-STAT3 pathway was involved in regulations of endogenous H2S for LPS-induced hepatocyte apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: β-Glucans have been shown to enhance immune responses for centuries, which contributes to their anti-tumor property. However, their mechanisms of action are still elusive. Dectin-1, the C-type lectin receptor for β-glucan, is expressed abundantly on dendritic cells (DCs). Activation of DCs via Dectin-1 can lead to the maturation of DC, inducing both innate and adaptive immune responses against tumor development and microbial infection. In this study, we found that particulate yeast-derived β-glucans could induce the maturation of murine dendritic cell line D2SC/1 cells and increase the expression of mGITRL on D2SC/1 cells via Dectin-1/Syk pathway in a dose dependent manner. Furthermore, we demonstrated that the increased mGITRL on D2SC/1 cells could impair the suppressive activity of CD4+CD25+ regulatory T cells (Tregs) and enhance the proliferation of CD4+CD25− effector T cells (Teffs). These findings suggest that particulate β-glucan can be used as immunomodulator to stimulate potent T cell-mediated adaptive immunity while down-regulate immune suppressive activity, leading to a more efficient defense mechanism against tumor development or infectious diseases. [Copyright &y& Elsevier]

Abstract: Apolipoprotein E (apoE) is an important plasma protein in cholesterol homeostasis and plays a key role in the progression of glomerulosclerosis (GS). We conducted this investigation to explore whether all-trans retinoic acid (ATRA) could regulate the apoE expression in the pathological process of GS. 120 Wistar rats were divided into three groups at random: sham operation group (SHO), glomerulosclerosis model group without treatment (GS), GS model group treated with ATRA (GA); n=40, respectively. The disease of GS in rat was established by uninephrectomy and adriamycin (5mg/kg) injection. At the end of 9 and 13weeks, 20 rats in each group were killed and the relevant samples were collected. 24-hour urine total protein (24UTP), 24-hour urine excretion for albumin (24Ualb), serum total protein (TP) and serum albumin (Alb), blood urea nitrogen (BUN), serum creatinine (Scr), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), serum and urine apoE and glomerulosclerosis index (GSI) were measured. The protein expressions of collagen IV (Col-IV), fibronectin (FN) and apoE in glomeruli were determined by immunohistochemistry. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was used to detect the expression of apoE mRNA in kidney. TP and Alb in GA group in 9/13-week were increased than those of GS group, however, the differences were not statistically significant. Compared with group GS at 9/13weeks, values of 24UTP, 24Ualb, BUN, Scr, TC, TG, HDL, LDL, serum and urine apoE, and GSI in GA group that were significantly reduced, and protein expressions of Col-IV, FN and apoE in glomeruli and expression of apoE mRNA in renal tissue were significantly down-regulated by ATRA (P <0.01). In conclusion, ATRA can regulate the expression of apoE, reduce the accumulation of extracellular matrix (ECM) and step down the progression of GS. [Copyright &y& Elsevier]