Prognostic factors in patients with bone marrow hemophagocytosis and its association with hematologic malignancies.

Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous group of hyperinflammatory statuses that are difficult to diagnose and can be life-threatening. Bone marrow (BM) hemophagocytosis is one of the diagnostic criteria according to HLH 2004 diagnostic criteria and HS score. Limited studies have focused on the prognostic factors of BM hemophagocytosis and its association with hematologic malignancies. We aimed to analyze the clinical significance of BM hemophagocytosis. Patients with BM hemophagocytosis, either by cytology or pathology, were enrolled at Taipei Veterans General Hospital from January 2002 to July 2021. Relevant clinical and laboratory data were extracted from medical records. Of 119 patients with BM hemophagocytosis, 57 were diagnosed with hematologic malignancies. The median age of the patients was 58, ranging from 21 to 90. Splenomegaly (adjusted odds ratio [aOR] 2.96; 95% confidence interval [CI] 1.13-7.79) was a risk factor for hematologic malignancies, while autoimmune disease (aOR 0.07; 95% CI 0.01-0.39) and increased D-dimer (aOR 0.25; 95% CI 0.07-0.92) were protective factors. Risk factors for mortality in patients with BM hemophagocytosis were hematologic malignancies (adjusted hazard ratio [aHR] 2.34; 95% CI 1.24-4.44), Eastern Cooperative Oncology Group score ≥3 (aHR 2.42; 95% CI 1.20-4.89) and thrombocytopenia (aHR 3.09; 95% CI 1.04-9.16). In conclusion, among patients with BM hemophagocytosis, splenomegaly was a predictor of hematologic malignancies. Patients with hematologic malignancies, poor performance status, or thrombocytopenia had a higher mortality risk. Further validation studies are warranted.

Etiology and Clinical Characteristics of Community-Acquired Pneumonia with Airway Malacia in Children.

Objective: The objective of this article is to study the etiology of community-acquired pneumonia in children with airway malacia. Methods: We retrospectively reviewed the medical records of 428 pneumonia patients. All patients underwent bronchoscopy, and bronchoalveolar lavage samples were processed for microbiological assessment. Results: In a total of 428 cases reviewed, 60 were found to have airway malacia. Pathogens were identified in 44 of the 60 specimens (73.3%), with 32 being single-pathogen infections. The most common pathogen was respiratory syncytial virus (RSV; 20%). Mixed-pathogen infections were observed in 12 patients. Airway malacia patients were younger than those without malacia (10.5 vs. 50 months, respectively; p < 0.001). Compared with those without airway malacia, wheezing, cyanosis and admission to the pediatric intensive care unit were more common in children with airway malacia and their hospital stay was longer. Conclusion: RSV was the most common pathogen in those with airway malacia. Airway malacia was found to aggravate infectious pneumonia.

MicroRNA-200 is induced by thioredoxin-interacting protein and regulates Zeb1 protein signaling and beta cell apoptosis.

Small noncoding microRNAs have emerged as important regulators of cellular processes, but their role in pancreatic beta cells has only started to be elucidated. Loss of pancreatic beta cells is a key factor in the pathogenesis of diabetes, and we have demonstrated that beta cell expression of thioredoxin-interacting protein (TXNIP) is increased in diabetes and causes beta cell apoptosis, whereas TXNIP deficiency is protective against diabetes. Recently, we found that TXNIP also impairs beta cell function by inducing microRNA (miR)-204. Interestingly, using INS-1 beta cells and primary islets, we have now discovered that expression of another microRNA, miR-200, is induced by TXNIP and by diabetes. Furthermore, we found that miR-200 targeted and decreased Zeb1 (zinc finger E-box-binding homeobox 1) and promoted beta cell apoptosis as measured by cleaved caspase-3 levels, Bax/Bcl2 ratio, and TUNEL. In addition, Zeb1 knockdown mimicked the miR-200 effects on beta cell apoptosis, suggesting that Zeb1 plays an important role in mediating miR-200 effects. Moreover, miR-200 increased beta cell expression of the epithelial marker E-cadherin, consistent with inhibition of epithelial-mesenchymal transition, a process thought to be involved in beta cell expansion. Thus, we have identified a novel TXNIP/miR-200/Zeb1/E-cadherin signaling pathway that, for the first time, links miR-200 to beta cell apoptosis and diabetes and also beta cell TXNIP to epithelial-mesenchymal transition. In addition, our results shed new light on the regulation and function of miR-200 in beta cells and show that TXNIP-induced microRNAs control various processes of beta cell biology.

Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2.

Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.

Characterization of calmodulin-Fas death domain interaction: an integrated experimental and computational study.

The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM-Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in lpr-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (Ka) for CaM-Fas DD binding from (1.79 ± 0.20) × 10(6) to (0.88 ± 0.14) × 10(6) M(-1) and slightly increased a standard state Gibbs free energy (ΔG°) for CaM-Fas DD binding from -8.87 ± 0.07 to -8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM-Fas DD interactions. Results from this study characterize CaM-Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM-Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM-Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis.

FOXO1 competes with carbohydrate response element-binding protein (ChREBP) and inhibits thioredoxin-interacting protein (TXNIP) transcription in pancreatic beta cells.

Thioredoxin-interacting protein (TXNIP) has emerged as an important factor in pancreatic beta cell biology, and tight regulation of TXNIP levels is necessary for beta cell survival. However, the mechanisms regulating TXNIP expression have only started to be elucidated. The forkhead boxO1 transcription factor (FOXO1) has been reported to up-regulate TXNIP expression in neurons and endothelial cells but to down-regulate TXNIP in liver, and the effects on beta cells have remained unknown. We now have found that FOXO1 binds to the TXNIP promoter in vivo in human islets and INS-1 beta cells and significantly decreases TXNIP expression. TXNIP promoter deletion analyses revealed that an E-box motif conferring carbohydrate response element-binding protein (ChREBP)-mediated, glucose-induced TXNIP expression is necessary and sufficient for this effect, and electromobility shift assays confirmed FOXO1 binding to this site. Moreover, FOXO1 blocked glucose-induced TXNIP expression and reduced glucose-induced ChREBP binding at the TXNIP promoter without affecting ChREBP expression or nuclear localization, suggesting that FOXO1 may compete with ChREBP for binding to the TXNIP promoter. In fact, a FOXO1 DNA-binding mutant (FOXO1-H215R) failed to inhibit TXNIP transcription, and the effects were not restricted to TXNIP as FOXO1 also inhibited transcription of other ChREBP target genes such as liver pyruvate kinase. Together, these results demonstrate that FOXO1 inhibits beta cell TXNIP transcription and suggest that FOXO1 confers this inhibition by interfering with ChREBP DNA binding at target gene promoters. Our findings thereby reveal a novel gene regulatory mechanism and a previously unappreciated cross-talk between FOXO1 and ChREBP, two major metabolic signaling pathways.

Hemp seeds attenuate loperamide-induced constipation in mice.

Constipation is a common gastrointestinal disease that seriously affects human physical and mental health. Studies have reported that hemp seeds can improve constipation, however the specific mechanism is still unclear. This study investigates that hemp seed (HS) and its water-ethanol extract (HSE) attenuates loperamide-induced constipation in mice. The research results show that: the fecal water content and small intestinal transit rate of mice in the hemp seed group and hemp seed hydroalcoholic extract group were significantly increased compared with MC group, and the first red feces defecation time was significantly shortened; HS and HSE significantly influence serum levels of Gastrin (Gas), motilin (MTL), substance P (SP), and endothelin (ET), potentially mediating their effects on gastrointestinal motility. HS and HSE can improve colon inflammation in constipated mice with H&E staining. Compared with the model of constipation group, the content of short-chain fatty acids in the HS group and HSE group increased significantly. Gut microbiome studies have shown that the structure and abundance of intestinal flora are altered. HS and HSE changed the abundance of Odoribacter, Bacteroide, Lactobacillus and Prevotella. Together, these results suggest that HS have the potential to stimulate the proliferation of beneficial gut microbes and promote intestinal motility, thereby improving gut health and relieving symptoms of constipation.

Calcium channel blockers act through nuclear factor Y to control transcription of key cardiac genes.

First-generation calcium channel blockers such as verapamil are a widely used class of antihypertensive drugs that block L-type calcium channels. We recently discovered that they also reduce cardiac expression of proapoptotic thioredoxin-interacting protein (TXNIP), suggesting that they may have unappreciated transcriptional effects. By use of TXNIP promoter deletion and mutation studies, we found that a CCAAT element was mediating verapamil-induced transcriptional repression and identified nuclear factor Y (NFY) to be the responsible transcription factor as assessed by overexpression/knockdown and luciferase and chromatin immunoprecipitation assays in cardiomyocytes and in vivo in diabetic mice receiving oral verapamil. We further discovered that increased NFY-DNA binding was associated with histone H4 deacetylation and transcriptional repression and mediated by inhibition of calcineurin signaling. It is noteworthy that the transcriptional control conferred by this newly identified verapamil-calcineurin-NFY signaling cascade was not limited to TXNIP, suggesting that it may modulate the expression of other NFY targets. Thus, verapamil induces a calcineurin-NFY signaling pathway that controls cardiac gene transcription and apoptosis and thereby may affect cardiac biology in previously unrecognized ways.

Calmodulin mediates Fas-induced FADD-independent survival signaling in pancreatic cancer cells via activation of Src-extracellular signal-regulated kinase (ERK).

Pancreatic cancer remains a devastating malignancy with a poor prognosis and is largely resistant to current therapies. To understand the resistance of pancreatic tumors to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. We found that knockdown of the Fas-associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation, rendered Fas-sensitive MiaPaCa-2 and BxPC-3 pancreatic cells resistant to Fas-induced apoptosis. By contrast, Fas activation promoted the survival of the FADD knockdown MiaPaCa-2 and BxPC-3 cells in a concentration-dependent manner. The pharmacological inhibitor of ERK, PD98059, abrogated Fas-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated to mediate Fas-induced ERK activation and cell survival. Immunoprecipitation of Fas in the FADD knockdown cells identified the presence of increased calmodulin, Src, and phosphorylated Src in the Fas-associated protein complex upon Fas activation. Trifluoperazine, a calmodulin antagonist, inhibited Fas-induced recruitment of calmodulin, Src, and phosphorylated Src. Consistently, trifluoperazine blocked Fas-promoted cell survival. A direct interaction of calmodulin and Src and their binding site were identified with recombinant proteins. These results support an essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors.

Reduced CaM/FLIP binding by a single point mutation in c-FLIP(L) modulates Fas-mediated apoptosis and decreases tumorigenesis.

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIP(L) in a Ca(2+)-dependent manner. Deletion of the CaM-binding region (amino acid 197-213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIP(L). Sequence analysis of the CaM-binding region at 197-213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIP(L) was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIP(L) binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIP(L) mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIP(L). Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIP(L) recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIP(L) binding to CaM, which mediates the anti-apoptotic function of c-FLIP(L), most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

Alpha Cell Thioredoxin-interacting Protein Deletion Improves Diabetes-associated Hyperglycemia and Hyperglucagonemia.

Thioredoxin-interacting protein (TXNIP) has emerged as a key factor in pancreatic beta cell biology, and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of TXNIP protects against diabetes in different mouse models. However, while TXNIP is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We generated an alpha cell TXNIP knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-induced diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed, and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus together the current studies suggest that downregulation of alpha cell TXNIP is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Tamoxifen enhances therapeutic effects of gemcitabine on cholangiocarcinoma tumorigenesis.

Cholangiocarcinoma is a highly malignant tumor with limited therapeutic options. We have previously reported that tamoxifen (TMX) induces apoptosis of cholangiocarcinoma cells and reduces cholangiocarcinoma tumorigenesis in mice. In the present studies, we determined the effect of combination therapy of TMX and gemcitabine (GMT), another chemotherapeutical reagent for many cancers, on cholangiocarcinoma tumorigenesis and investigated the responsible mechanisms. GMT inhibited cell growth and induced apoptosis of cholangiocarcinoma cells in a concentration-dependent manner. TMX enhanced GMT-induced apoptosis of cholangiocarcinoma cells. Consistently, GMT (15 mg/kg) inhibited cholangiocarcinoma tumorigenesis in nude mice by 50%. TMX (15 mg/kg) enhanced the inhibitory effect of GMT on tumorigenesis by 33%. The inhibition of tumor growth correlated with enhanced apoptosis in tumor tissues. To elucidate the mechanisms underlying the additive effects of TMX on GMT-induced apoptosis, we determined the activation of caspases in cholangiocarcinoma cells exposed to GMT, TMX, or both. Activation of caspases 9 and 3, as well as cytochrome c release to the cytosol, was demonstrated in cells exposed to both reagents. In contrast, TMX activated caspase 2, whereas GMT had no effect. Inhibition of caspase 2 activation decreased TMX-, but not GMT-, induced activation of caspase 3 and apoptosis of cholangiocarcinoma cells. Similarly, activation of caspase 2 was found in tumors from TMX-treated mice, but not GMT-treated mice. Therefore, the enhanced effect of TMX on GMT-induced cholangiocarcinoma cell death is partially mediated by activation of caspase 2. TMX and GMT both induce apoptosis and inhibit cholangiocarcinoma tumorigenesis, which may be attributed to the activation of distinct apoptosis signals by TMX and GMT. Our studies provide in vivo evidence and molecular insight to support the use of TMX and GMT in combination as an effective therapy for cholangiocarcinoma.

Human Glucagon Expression Is under the Control of miR-320a.

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.

Identification of an Anti-diabetic, Orally Available Small Molecule that Regulates TXNIP Expression and Glucagon Action.

Diabetes is characterized by hyperglycemia, loss of functional islet beta cell mass, deficiency of glucose-lowering insulin, and persistent alpha cell secretion of gluconeogenic glucagon. Still, no therapies that target these underlying processes are available. We therefore performed high-throughput screening of 300,000 compounds and extensive medicinal chemistry optimization and here report the discovery of SRI-37330, an orally bioavailable, non-toxic small molecule, which effectively rescued mice from streptozotocin- and obesity-induced (db/db) diabetes. Interestingly, in rat cells and in mouse and human islets, SRI-37330 inhibited expression and signaling of thioredoxin-interacting protein, which we have previously found to be elevated in diabetes and to have detrimental effects on islet function. In addition, SRI-37330 treatment inhibited glucagon secretion and function, reduced hepatic glucose production, and reversed hepatic steatosis. Thus, these studies describe a newly designed chemical compound that, compared to currently available therapies, may provide a distinct and effective approach to treating diabetes.

Immunolocalization of an acid phosphatase from pearl oyster (Pinctada fucata) and its in vitro effects on calcium carbonate crystal formation.

Distribution of an acid phosphatase, AcPase I, from pearl oyster (Pinctada fucata) in different tissues was investigated via enzyme activity determination and immunohistochemistry. Positive reactions were observed in sections of digestive gland, base of gill filaments, and epithelia of the outer side of the middle fold and the inner side of the outer fold, which indicated AcPase I might participate in processes besides immune defense, such as calcium metabolism or shell formation. Its effects on CaCO(3) crystal formation were studied in vitro. Results revealed that AcPase I inhibited CaCO(3) precipitation in a dose-dependent manner and had no affinity for calcium. CaCO(3) crystals induced by AcPase I exhibited a cluster needle-like morphology, which proved to be aragonite. The morphology and size of the aragonites varied with different concentrations of AcPase I. Our observations described here may provide important clues to further understanding of the correlations between mineralization and immune defense in the oyster.

Different effects of Pb(2+) and Cu(2+) on immune and antioxidant enzyme activities in the mantle of Pinctada fucata.

The aim of this study was to investigate the natural role of the mantle in pearl oyster, Pinctada fucata. The mantle is believed to be the tissue responsible for shell and pearl formation. However, our current study on lead and copper accumulation in tissues of the oyster showed that the secondary tissue for lead accumulation was not the digestive gland but the mantle. In view of high lead concentrations in the mantle, its general metabolic condition (including immune and antioxidant defense systems) as affected by the two metals was studied. The results indicated that activities of antioxidant enzymes (superoxide dismutase, SOD; Se-dependent glutathione peroxidase, Se-GPx) were altered by lead and copper in the similar way. However, the immune enzyme activities (acid phosphatase, AcPase; phenoloxidase, PO) were perturbed differently by two metals. Therefore, the mantle of P. fucata was predicted to participate in immune processes and accumulation or detoxification of lead besides shell formation. Our observations described here may also provide important clues to further understanding of the biomarker responses of bivalves.

Purification and partial characterization of two acid phosphatase forms from pearl oyster (Pinctada fucata).

The present study describes the details about the acid phosphatase forms in the pearl oyster, Pinctada fucata. Two isoenzymes (AcPase I and II) of acid phosphatase were separated and purified from viscera of pearl oyster, P. fucata to homogeneity by chromatography on DEAE-Sepharose Fast Flow, Sephadex G-200 superfine and ConA Sepharose 4B, and partial biochemical properties of AcPase I and II were studied. AcPase I and AcPase II had molecular weights of 208.8 and 64.3 kDa, respectively. AcPase I was a single polypeptide chain, while AcPase II was a dimeric enzyme composed of two equivalent subunits. AcPase I and II showed optimal pHs at 4.6 and 3.2 with p-nitrophenylphosphate as substrate. The optimal catalytic reaction temperature was 47 degrees C for AcPase I and 57 degrees C for AcPase II. Both enzyme forms were stable when incubated at 50 degrees C for 40 min. Tartrate and fluoride were the most effective inhibitors of the enzymes. Fe(3+), Zn(2+), Cu(2+) and Pb(2+) inhibited the activity of AcPase I and II to differing extents. AcPase I and II were apparently nonspecific and hydrolyzed various phosphoric esters. The different properties of AcPase I and II suggested that the two enzymes may play different roles in the pearl oyster.

Islet ChREBP-ß is increased in diabetes and controls ChREBP-α and glucose-induced gene expression via a negative feedback loop.

OBJECTIVE: Carbohydrate-response element-binding protein (ChREBP) is the major transcription factor conferring glucose-induced gene expression in pancreatic islets, liver and adipose tissue. Recently, a novel ChREBP isoform, ChREBP-ß, was identified in adipose tissue and found to be also expressed in islets and involved in glucose-induced beta cell proliferation. However, the physiological function of this less abundant ß-isoform in the islet, and in diabetes, is largely unknown. The aims of the present study, therefore, were to determine how diabetes affects ChREBP-ß and elucidate its physiological role in pancreatic beta cells. METHODS: Non-obese diabetic and obese, diabetic ob/ob mice were used as models of T1D and T2D and human islets and the rat INS-1 beta cell line were exposed to low/high glucose and used for ChREBP isoform-specific gain-and-loss-of-function experiments. Changes in ChREBP-ß and ChREBP-α were assessed by qRT-PCR, immunoblotting, promoter luciferase, and chromatin immunoprecipitation studies. RESULTS: Expression of the ChREBP-ß isoform was highly induced in diabetes and by glucose, whereas ChREBP-α was downregulated. Interestingly, ChREBP-ß gain-of-function experiments further revealed that it was ChREBP-ß that downregulated ChREBP-α through a negative feedback loop. On the other hand, ChREBP-ß knockdown led to unabated ChREBP-α activity and glucose-induced expression of target genes, suggesting that one of the physiological roles of this novel ß-isoform is to help keep glucose-induced and ChREBP-α-mediated gene expression under control. CONCLUSIONS: We have identified a previously unappreciated negative feedback loop by which glucose-induced ChREBP-ß downregulates ChREBP-α-signaling providing new insight into the physiological role of islet ChREBP-ß and into the regulation of glucose-induced gene expression.

miR-204 Targets PERK and Regulates UPR Signaling and ß-Cell Apoptosis.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of diabetes and the associated ß-cell apoptosis. Although microRNAs (miRNAs) have been widely studied in various diseases including diabetes, the role of miRNAs in ER stress and ß-cell apoptosis has only started to be elucidated. We recently showed that diabetes increases ß-cell miR-204 and have now discovered that miR-204 directly targets the 3'untranslated region of protein kinase R-like ER kinase (PERK), 1 of the 3 ER transmembrane sensors and a key factor of the unfolded protein response (UPR). In addition, by using primary human islets, mouse islets, and INS-1 ß-cells, we found that miR-204 decreased PERK expression as well as its downstream factors, activating transcription factor 4 and CCAAT enhancer-binding protein homologous protein, whereas it had no effect on the other 2 ER transmembrane sensors, activating transcription factor 6 and inositol-requiring enzyme-1α. Interestingly, we discovered that miR-204 also inhibited PERK signaling in the context of ER stress, and this exacerbated ER stress-induced ß-cell apoptosis. This effect could be mimicked by PERK inhibitors supporting the notion that the miR-204-mediated inhibition of PERK and UPR signaling was conferring these detrimental effects on cell survival. Taken together, we have identified PERK as a novel target of miR-204 and show that miR-204 inhibits PERK signaling and increases ER stress-induced cell death, revealing for the first time a link between this miRNA and UPR.

[Effect of high performance liquid chromatographic instrument system on the analysis of erythromycin A oxime].

A HPLC chromatography for the determination of erythromycin A oxime and relative compounds was studied, and the effect of chromatography systems including a HITACHI L-7100, a Shimadzu LC-6A, a Waters 474 and relative columns was analyzed. It was revealed that different HPLC apparatus and columns have obvious impact on the peak separation and retention time under the general chromatographic condition. The suitable chromatographic conditions for several different chromatography systems were summarized with good linear relationship, which is very significant to the quality control of erythromycin A oxime and relative compounds.