Endogenous prostaglandins (PGs) are involved in adaptive gastric protection against acute injury, and cyclooxygenase (COX)-1 is responsible for the production of PGs in this phenomenon. In the present study, we examined the effect of various COX inhibitors on gastric ulcerogenic and acid secretory responses following daily exposure of the stomach to iodoacetamide (IA) and investigated the role for COX isozyme in gastric protection under subchronic mucosal irritation. Gastric mucosal irritation was induced by addition of 0.1% IA to drinking water, and the gastric mucosa was examined on the 6th day. Indomethacin (5 mg/kg) or SC-560 (selective COX-1 inhibitor, 5 mg/kg) or rofecoxib (selective COX-2 inhibitor, 5 mg/kg) was given p.o. twice 24 hr and 3 hr before the termination of IA treatment. Giving IA in drinking water for 5 days produced minimal damage in the stomach. The damage was significantly worsened by indomethacin, resulting in hemorrhagic lesions. Both SC-560 and rofecoxib also aggravated such lesions, although the effect of rofecoxib was more pronounced. Treatment with IA decreased acid secretion in pylorus-ligated stomachs, and this change was significantly reverted by indomethacin as well as SC-560 and rofecoxib. Mucosal PGE2 content was increased following IA treatment, with apparent expression of COX-2 mRNA in the stomach, and the increased PGE2 production was significantly suppressed by SC-560 and rofecoxib as well as indomethacin. These results suggest that endogenous PGs derived from both COX-1 and COX-2 are involved in the mucosal defense of the inflamed stomach, partly by decreasing acid secretion and contribute to maintaining the mucosal integrity under such conditions.
Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.
Experimental liver disorders were induced by the use of carbon tetrachloride or D-galactosamine hydrochloride in rats maintained on a vitamin E deficient diet and in rats fed a diet supplemented with vitamin E, and the protective effect of vitamin E on the liver was determined. After exposure to carbon tetrachloride or D-galactosamine hydrochloride the serum levels of transaminases, lysosomal enzyme beta-glucuronidase, and acid phosphatase were elevated, and thiobarbituric acid reactive substances in serum and liver homogenate were also increased. The changes were conspicuous in the vitamin E deficient rats, but were only slight in rats fed a diet supplemented with vitamin E. The results of this study suggest that vitamin E has a protective effect on liver disorders by inhibiting lysosomal enzyme liberation and lipid peroxidation.
Abstract The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization‐induced neurite outgrowth and the expression of growth‐associated protein‐43 (GAP‐43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP‐43 and synapsin I. These increases were suppressed by inhibition of Ca 2+ ‐calmodulin‐dependent protein kinase II (CaMKII), PLD, or mitogen‐activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization‐induced neurite outgrowth, and the increase in GAP‐43 and synapsin I expression. Depolarization evoked a Ca 2+ rise that activated various signaling enzymes and the cAMP response element‐binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl‐induced phosphorylation of proline‐rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal‐regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1‐butanol and transfection of PLD2 siRNA, whereas it was enhanced by over‐expression of wild‐type PLD2. Depolarization‐induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization‐induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP‐43 and synapsin I expression during depolarization‐induced neuronal differentiation in PC12 cells.
Carnitine is a vitamin-like compound that plays important roles in fatty acid β-oxidation and the control of the mitochondrial coenzyme A/acetyl-CoA ratio. However, carnitine is not added to ordinary enteral nutrition or total parenteral nutrition. In this study, we determined the serum carnitine concentrations in subjects receiving ordinary enteral nutrition (EN) or total parenteral nutrition (TPN) and in patients with inflammatory bowel diseases to compare its levels with those of other nutritional markers. Serum samples obtained from 11 EN and 11 TPN patients and 82 healthy controls were examined. In addition, 10 Crohn's disease and 10 ulcerative colitis patients with malnutrition who were barely able to ingest an ordinary diet were also evaluated. Carnitine and its derivatives were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carnitine concentrations in EN and TPN subjects were significantly lower compared with those of the control subjects. Neither the serum albumin nor the total cholesterol level was correlated with the carnitine concentration, although a significant positive correlation was found between the serum albumin and total cholesterol levels. Indeed, patients with CD and UC showed significantly reduced serum albumin and/or total cholesterol levels, but their carnitine concentrations remained normal. In conclusion, only a complete blockade of an ordinary diet, such as EN or TPN, caused a reduction in the serum carnitine concentration. Serum carnitine may be an independent biomarker of malnutrition, and its supplementation is needed in EN and TPN subjects even if their serum albumin and total cholesterol levels are normal.
Background: Milademetan (DS-3032b) is a small-molecule MDM2 inhibitor that activates p53 and induces apoptosis by disrupting the MDM2-p53 interaction. Milademetan has demonstrated single-agent antileukemic activity in preclinical and clinical studies in AML. AZA, a hypomethylating agent, is used to treat AML and myelodysplastic syndromes by upregulating epigenetically silenced genes in AML cells. We conducted a preclinical study of milademetan in combination with AZA because both agents modulate gene expression related to cell growth or apoptosis. Aims: To investigate the efficacy of milademetan in combination with AZA in TP53 wild-type AML and to identify the underlying molecular mechanisms of action. Methods: The effects of milademetan and AZA as single agents and in combination were assessed in vitro and in vivo. The human TP53 wild-type AML cell lines MOLM-13, ML-2, and OCI-AML5 were investigated. Cell growth was evaluated in ATP assays, and apoptosis induction was assessed in annexin V assays. In vivo activity was evaluated in mice that were subcutaneously xenografted with MOLM-13 cells then treated with oral milademetan for 7 days and intravenous AZA for 5 days. Results: The growth inhibitory effects of milademetan and AZA in various treatment schedules as single agents and in combination were investigated in AML cell lines and in vivo using the MOLM-13 xenograft model. In the xenograft study, a sequential combination of AZA followed by milademetan caused greater tumor growth inhibition than single-agent and other combination schedules of milademetan and AZA. This sequential combination increased annexin V–positive cells and upregulated p53 and p21 protein expression in cell lines. Interestingly, AZA treatment induced a protein that may be a faster-migrating form of MDM2. Characterization of this protein and further analyses, including RNA sequencing and genome-wide DNA methylation profiling, to provide additional mechanistic insights are ongoing. Summary/Conclusion: Preclinical results demonstrated that a sequential combination treatment with AZA followed by milademetan enhanced antileukemic activity in cell lines and a mouse xenograft model. A phase 1 clinical trial of this combination has been initiated based on these results.
