Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 μM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1–2 μM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. B. Ozpolat and U. Akar contributed equally.     

Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+‐transporters in heart failure

Zn²⁺‐homoeostasis including free Zn²⁺ ([Zn²⁺]_i) is regulated through Zn²⁺‐transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn²⁺‐transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn²⁺]_i was significantly high in doxorubicin‐treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα‐phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn²⁺]_i using zinc‐ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn²⁺ transporters. Additionally, increased [Zn²⁺]_i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn²⁺]_i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn²⁺ transporters on an intersection pathway with increased [Zn²⁺]_i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well‐controlled [Zn²⁺]_i via Zn²⁺ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF.     

Patterns of genetic diversity and linkage disequilibrium in a highly structured Hordeum vulgare association-mapping population for the Mediterranean basin

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT^® markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r ² of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT^® markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.     

Comparison of different needle diameters and flow rates on bone marrow mononuclear stem cell viability: an ex vivo experimental study

Considerable information is available regarding the experimental and clinical applications of bone marrow derived stem cells (BMDSC) for regenerative medicine. Optimized stem cell delivery systems might help to maintain better stem cell viability. We have investigated whether needle diameters and flow rates through the needles cause any difference in terms of BMDSC viability.     

Biosorption of a textile dye (Acid Blue 40) by cone biomass of Thuja orientalis: estimation of equilibrium, thermodynamic and kinetic parameters

Biosorption of Acid Blue 40 (AB40) onto cone biomass of Thuja orientalis was studied with variation in the parameters of pH, contact time, biosorbent and dye concentration and temperature to estimate the equilibrium, thermodynamic and kinetic parameters. The AB40 biosorption was fast and the equilibrium was attained within 50 min. Equilibrium data fitted well to the Langmuir isotherm model in the studied concentration range of AB40 and at various temperatures. Maximum biosorption capacity (q(max)) for AB40 was 2.05 x 10(-4)mol g(-1) or 97.06 mg g(-1) at 20 degrees C. The changes of Gibbs free energy, enthalpy and entropy of biosorption were also evaluated for the biosorption of AB40 onto T. orientalis. The results indicate that the biosorption was spontaneous and exothermic. Kinetics of biosorption of AB40 was analyzed and rate constants were also derived and the results show that the pseudo-second-order kinetic model agrees very well with the experimental data.     

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta

Little is knownabout the function and regulation of theNa⁺-K⁺-2Clcotransporter NKCC1 in vascular smooth muscle. Theactivity of NKCC1 was measured as the bumetanide-sensitive efflux of⁸⁶Rb⁺from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH₂O) rapidlydoubled cotransporter activity, consistent with its volume-regulatoryfunction. NKCC1 was also acutely activated by the vasoconstrictors ANGII (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl(54%). Both nitric oxide and nitroprusside inhibited basal NKCC1activity (39 and 34%, respectively), and nitroprussidecompletely reversed the stimulation by phenylephrine. Thephosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced byphenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. Weconclude that theNa⁺-K⁺-2Clcotransporter in vascular smooth muscle is reciprocally regulated byvasoconstrictors and nitrovasodilators and contributes to smooth musclecontraction, indicating that alterations in NKCC1 could influencevascular smooth muscle tone in vivo.

     

Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.     

Tissue transglutaminase inhibits autophagy in pancreatic cancer cells

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.     

Preparation of pH- and ionic-strength responsive biodegradable fumaric acid crosslinked carboxymethyl cellulose

A novel biodegradable sodium carboxymethyl cellulose (NaCMC)-based hydrogel was synthesized by using fumaric acid (FA) as a crosslinking agent at various ratios. Hydrogels (CMCF) were characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Swelling behaviors of hydrogels were investigated in distilled water, various salt, and pH solutions. The FTIR results indicated the crosslinking between carboxyl groups of FA with hydroxyl group of NaCMC through ester formation. AFM analyses showed that roughness of hydrogel surface decreased with increasing crosslinker concentration. The swelling capacity decreased with an increase in charge of the metal cation (Al(3+)相似文献