Histidine re-sensitizes pediatric acute lymphoblastic leukemia to 6-mercaptopurine through tetrahydrofolate consumption and SIRT5-mediated desuccinylation.

Despite progressive improvements in the survival rate of pediatric B-cell lineage acute lymphoblastic leukemia (B-ALL), chemoresistance-induced disease progression and recurrence still occur with poor prognosis, thus highlighting the urgent need to eradicate drug resistance in B-ALL. The 6-mercaptopurine (6-MP) is the backbone of ALL combination chemotherapy, and resistance to it is crucially related to relapse. The present study couples chemoresistance in pediatric B-ALL with histidine metabolism deficiency. Evidence was provided that histidine supplementation significantly shifts the 6-MP dose-response in 6-MP-resistant B-ALL. It is revealed that increased tetrahydrofolate consumption via histidine catabolism partially explains the re-sensitization ability of histidine. More importantly, this work provides fresh insights into that desuccinylation mediated by SIRT5 is an indispensable and synergistic requirement for histidine combination therapy against 6-MP resistance, which is undisclosed previously and demonstrates a rational strategy to ameliorate chemoresistance and protect pediatric patients with B-ALL from disease progression or relapse.

Effect of Corilagin on the Proliferation and NF-κB in U251 Glioblastoma Cells and U251 Glioblastoma Stem-Like Cells.

Background. This study is to explore the effect of corilagin on the proliferation and NF-κB signaling pathway in U251 glioblastoma cells and U251 glioblastoma stem-like cells. Methods. CD133 positive U251 glioblastoma cells were separated by immunomagnetic beads to isolate glioblastoma stem-like cells. U251 cells and stem-like cells were intervened by different corilagin concentrations (0, 25, 50, and 100 µg/mL) for 48 h, respectively. Cell morphology, cell counting kit-8 assay, flow cytometry, dual luciferase reporter assay, and a western blot were used to detect and analyze the cell proliferation and cell cycle and investigate the expression of IKBα protein in cytoplasm and NF-κB/p65 in nucleus. Results. Corilagin inhibited the cell proliferation of U251 cells and their stem-like cells and the inhibition role was stronger in U251 stem-like cells (P < 0.05). The cell cycle was arrested at G2/M phase in the U251 cells following corilagin intervention; the proportion of cells in G2/M phase increased as the concentration of corilagin increased (P < 0.05). The U251 stem-like cells were arrested at the S phase following treatment with corilagin; the proportion of cells in the S phase increased as the concentration of corilagin increased (P < 0.05). The ratio of dual luciferase activities of U251 stem-like cells was lower than that of U251 cells in the same corilagin concentration. With increasing concentrations of corilagin, the IKBα expression in cytoplasm of U251 cells and U251 stem-like cells was increased, but the p65 expression in nucleus of U251 cells and U251 stem-like cells was decreased (P < 0.05). Conclusion. Corilagin can inhibit the proliferation of glioblastoma cells and glioblastoma stem-like cells; the inhibition on glioblastoma stem-like cell proliferation is stronger than glioblastoma cells. This different result indicates that the effect of corilagin on U251 cells and U251 stem-like cells may have close relationships with mechanism of cell cycle and NF-κB signaling pathway; however, the real antitumor mechanism of corilagin is not yet clear and requires further study.

Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco.

The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3-A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the ß-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance.

[Value of muscle enzyme analysis in differential diagnosis of childhood myopathic hyper-creatine kinase-emia].

OBJECTIVE: To summarize the etiology and clinical characteristics of children with myopathic elevated creatine kinase (CK) levels. The degrees of elevated CK as well as lactic dehydrogenase (LDH) and aspartate aminotransferase (AST) levels in different myopathy were analyzed. METHODS: The clinical data of 235 cases characterized as myopathic hyper-CK-emia from January 2004 to December 2011 were collected and analyzed. A retrospective analysis of LDH and AST levels according to CK in part of the patients were reviewed. RESULTS: Of the 235 cases, 180 were male and 55 female. According to the age at which hyper-CK-emia was diagnosed, 64 cases were under 6 months, 90 between 6 months and 3 years, 50 between 3 and 6 years and 31 between 6 and 14 years. Their CK levels significantly increased in 162 cases, moderately increased in 31 cases, and slightly increased in 42 cases. The age at which hyper-CK-emia was diagnosed and the CK level had no correlation with muscle weakness and the severity. As to CK levels: Duchenne muscular dystrophy (DMD) > inflammatory myopathies > congenital muscular dystrophy (CMD) > metabolic myopathies. LDH and AST levels: DMD > inflammatory myopathies > metabolic myopathies > CMD. CONCLUSION: Unlike adults, the etiology of myopathic hyper-CK-emia in children is complicated and diverse. The onset type, the degree and duration of hyper-CK-emia are helpful to make the diagnosis. CK increases most significantly in DMD, then in inflammatory myopathies, CMD, and metabolic myopathies. Diagnostic flowchart of myogenic hyper-CK-emia should follow a certain process, and the indications of biochemical tests, metabolic screening, electrophysiological examination, muscle biopsy and genetic testing should be made. Finally, different treatments should be designed according to the etiology.

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression.

Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic ß-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the ß5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant.

Melanoma differentiation-associated gene 5 (MDA5) is one of the three members in the retinoic acid-inducible gene I-like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up-regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over-expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co-transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion.

Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18α-glycyrrhetinic acid (18α-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

Activated protein C can be used as a prophylactic as well as a therapeutic agent for heat stroke in rodents.

The present study was attempted to assess the prophylactic and the therapeutic effect of human recombinant activated protein C (APC; drotrecogin-alpha, activated) in experimental heat stroke. Anesthetized rats were divided into two groups and given vehicle solution 1 h before the start or immediately after the termination of heat stress (isotonic sodium chloride solution, 2 mL kg(-1) of body weight, i.v.) or APC (1-10 mg in 2 mL of isotonic sodium chloride solution per kilogram of body weight, i.v.). They were exposed to ambient temperature of 40 degrees C for 100 min to induce heat stroke. When the vehicle-pretreated rats underwent heat stress, their survival time values were found to be 57 to 71 min. Pretreatment or treatment with APC significantly increased survival time (122-221 min). All vehicle-pretreated heat stroke animals displayed systemic inflammation (evidenced by increased TNF-alpha, IL-1alpha, and IL-6) and activated coagulation (evidenced by increased levels of activated partial thromboplastin time, prothrombin time, and D-dimer and decreased levels of both platelet count and protein C). Biochemical assay also revealed that both renal and hepatic dysfunction (e.g., increased plasma levels of blood urea nitrogen, creatinine, adenine aminotransferase, aspartate aminotransferase, and alkaline phosphatase) were noted during heat stroke. A significant decrease in both cerebral blood flow and partial pressure of oxygen in hypothalamus were also observed in vehicle-pretreated heat stroke animals. These heat stroke reactions were all significantly reduced by pretreatment or treatment with human recombinant APC. The results indicate that human recombinant APC can be used as a prophylactic and a therapeutic agent for experimental heat stroke by ameliorating systemic inflammation, hypercoagulable state, and multiple organ dysfunction.

Effects of nitric oxide and hydrogen sulfide on the relaxation of pulmonary arteries in rats.

BACKGROUND: The balance between vasodilation and vasoconstriction plays a major role in maintaining vascular homeostasis. However, the underlying mechanisms are unclear. More and more evidence suggested that there was an interaction in the regulation of vasorelaxation between nitric oxide (NO) and hydrogen sulfide (H(2)S). We explored the interaction between and effects of NO and H(2)S on the relaxation of pulmonary arteries in rats. METHODS: Seven male Sprague-Dawley rats were anaesthetized with chloral hydrate and the pulmonary arteries of each rat separated for the study of vascular activities. The vasorelaxing activities of pulmonary artery rings in response to different doses of a NO donor, sodium nitroprusside (SNP), or a H(2)S donor, sodium hydrogen sulfide (NaHS), were measured in vitro. When pulmonary artery rings were treated with a cystathionine-gamma-lyase inhibitor, DL-propargylglycine, in the presence of SNP or a nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, in the presence of NaHS, the changes in relaxing activities were analyzed. RESULTS: The relaxation of pulmonary artery rings was in a dose dependent manner in response to either SNP or NaHS. The relaxation rates of pulmonary artery rings increased from (30.90+/-4.62)% to (60.50+/-8.08)% when the concentration of SNP increased from 1 micromol/L to 3 micromol/L and from (26.13+/-4.12)% to (53.09+/-14.01)% when the concentration of NaHS increased from 25 micromol/L to 100 micromol/L. However, when appropriate inhibitor was added, the relaxation responses to SNP and NaHS decreased. CONCLUSIONS: The results suggested that similarly to NO, H(2)S acted as a vasorelaxant either independently of, or synergistically with NO in the regulation of vasorelaxation. The interaction between NO and H(2)S played an important role in regulating relaxing activities of pulmonary arteries.