Neutral sphingomyelinase-3 is a DNA damage and nongenotoxic stress-regulated gene that is deregulated in human malignancies

In this study, we report the characterization of a novel genotoxic and nongenotoxic stress-regulated gene that we had previously named as SKNY. Our results indicate that SKNY encodes the recently identified neutral sphingomyelinase-3 (nSMase3; hereafter SKNY is referred to as nSMase3). Examination of nSMase3 subcellular distribution reveals nSMase3 to localize to the endoplasmic reticulum (ER), and deletion of a COOH-terminal region containing its putative transmembrane domain and ER targeting signal partly alters its compartmentalization to the ER. Treatment with genotoxic Adriamycin and nongenotoxic tumor necrosis factor-alpha up-regulates endogenous nSMase3 expression, albeit with different kinetics. Tumor necrosis factor-alpha up-regulates nSMase3 expression within 2 h that lasts beyond 24 h and declines to control levels by 36 h. Adriamycin up-regulation of nSMase3 is transient, occurs within 30 min, and declines to control levels by 120 min. Prolonged treatment with Adriamycin by 24 h and beyond, however, causes a down-regulation in nSMase3 expression. Activation of wild-type p53 also down-regulates nSMase3 expression, suggesting that DNA damage-mediated nSMase3 down-regulation seems to occur partly through the tumor suppressor p53. Overexpression of exogenous nSMase3 sensitizes cells to Adriamycin-induced cell killing, a finding consistent with the proposed proapoptotic role of nSMase enzymes and nSMase-generated ceramide. We further investigated nSMase3 expression in various human malignancies and found its expression to be deregulated in several types of primary tumors when compared with their matching normal tissues. Collectively, our results have identified nSMase3 to be an important molecule that is linked to tumorigenesis and cellular stress response.     

Tissue Selective Androgen Receptor Modulators (SARMs) Increase Pelvic Floor Muscle Mass in Ovariectomized Mice

Stress urinary incontinence (SUI), a prevalent condition, is represented by an involuntary leakage of urine that results, at least in part, from weakened or damaged pelvic floor muscles and is triggered by physical stress. Current treatment options are limited with no oral therapies available. The pelvic floor is rich in androgen receptor and molecules with anabolic activity including selective androgen receptor modulators (SARMs) may serve as therapeutic options for individuals with SUI. In this study, two SARMs (GTx‐024 and GTx‐027) were evaluated in a post‐menopausal animal model in order to determine their effect on pelvic floor muscles. Female C57BL/6 mice were ovariectomized and their pelvic muscles allowed to regress. The animals were then treated with vehicle or doses of GTx‐024 or GTx‐027. Animal total body weight, lean body mass, and pelvic floor muscle weights were measured along with the expression of genes associated with muscle catabolism. Treatment with the SARMs resulted in a restoration of the pelvic muscles to the sham‐operated weight. Coordinately, the induction of genes associated with muscle catabolism was inhibited. Although a trend was observed towards an increase in total lean body mass in the SARM‐treated groups, no significant differences were detected. Treatment of an ovariectomized mouse model with SARMs resulted in an increase in pelvic floor muscles, which may translate to an improvement of symptoms associated with SUI and serves as the basis for evaluating their clinical use. J. Cell. Biochem. 118: 640–646, 2017. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Alteration of ceramide synthase 6/C16-ceramide induces activating transcription factor 6-mediated endoplasmic reticulum (ER) stress and apoptosis via perturbation of cellular Ca2+ and ER/Golgi membrane network

Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1-6 (CerS1-6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C(16)-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C(16)-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca(2+) from the ER stores ([Ca(2+)](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C(16)-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca(2+) chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C(16)-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C(16)-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas.     

An alkaline phosphatase/phosphodiesterase, PhoD, induced by salt stress and secreted out of the cells of Aphanothece halophytica, a halotolerant cyanobacterium

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the phoD gene from a halotolerant cyanobacterium, Aphanothece halophytica (phoD(Ap)). The deduced protein, PhoD(Ap), contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD(Ap) enzyme was activated by Ca(2+) and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD(Ap) in a transformed cyanobacterium. Expression of the phoD(Ap) gene in A. halophytica cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that A. halophytica cells possess a PhoD that participates in the assimilation of P under salinity stress.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

Shoot meristem culture eliminates bacterial and fungal infections from elite varieties of turmeric (Curcuma longa L.)

In Vitro Cellular & Developmental Biology - Plant - Turmeric (Curcuma longa L. (Zingiberaceae)) is a rich source of medicinally important chemical compounds obtained from both pseudostem...     

Rapid shortening of telomere length in response to ceramide involves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase

Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.     

Clinical severity of β-thalassaemia/Hb E disease is associated with differential activities of the calpain-calpastatin proteolytic system

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in β-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in β-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of β(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of μ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.     

Screening sugarcane (Saccharum sp.) genotypes for salt tolerance using multivariate cluster analysis

Disease-free sugarcane plantlets of 11 cultivars derived from meristem cuttings were photoautotrophically grown on the MS medium and subsequently exposed to 0 (control) or 200?mM NaCl (salt stress) for 14?days. Sodium ion (Na⁺) in all sugarcane varieties was enriched when plantlets were subjected to 200?mM NaCl, except K88-1. Chlorophyll a (Chl_a), chlorophyll b (Chl_b) and total carotenoids (C_x+c), in the salt stressed leaves of all genotypes decreased significantly, but the extent of decrease was variable among different genotypes. In contrast, proline content in salt stressed plantlets of all sugarcane genotypes increased markedly, except in genotypes K95, K92-2 and LK92-4. Maximum quantum yield of PSII (F_v/F_m), photon yield of PSII (??_PSII), quantum efficiency of PSII (qP) and net photosynthetic rate (P_n) in salt stressed plantlets of all genotypes were significantly dropped, whereas ??_PSII and qP in cv. KK88-1 were alleviated, resulting in improved P_n. Moreover, growth parameters including shoot height, root length, fresh weight, dry weight and leaf area in salt stressed plantlets of all genotypes were significantly inhibited. The Na⁺ accumulation, pigment degradation, proline accumulation, photosynthetic abilities and growth inhibition in saline regimes were subjected to Hierarchical cluster analysis. Salt tolerant, K88-1 and UT94-7 and salt susceptible, K92-2 and LK92-4 classes of sugarcane genotypes were classified. The salt tolerant cultivars may be further studied including yield, sugar content and ratoon recovery rate in saline field trials.