Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression.

BACKGROUND: The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. RESULTS: Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. CONCLUSIONS: The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

Isoliquiritigenin, from Dalbergia odorifera, up-regulates anti-inflammatory heme oxygenase-1 expression in RAW264.7 macrophages.

OBJECTIVES: Isoliquiritigenin (ISL), one of the major constituents of Dalbergia odorifera T. Chen (Leguminosae), is reported to exert anti-inflammatory effects, but the relevant anti-inflammatory mechanisms are not completely understood. Heme oxygenase-1 (HO-1) has been proven to be involved in the resolution of inflammatory responses. In this study, we investigated whether ISL could induce HO-1 expression in RAW264.7 macrophages, and if so, whether HO-1 could mediate the anti-inflammatory effects of ISL. METHODS: The protein expression of inducible nitric oxide synthase and HO-1 was analyzed by western blot analysis. The production of nitric oxide (NO) and interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was assayed by Griess and ELISA, respectively. The TNF-alpha and HO-1 mRNA expression was analyzed by northern blot analysis. RESULTS: ISL markedly suppressed LPS-induced NO, IL-1beta, and TNF-alpha production. ISL induced HO-1 expression through the extracellular signal-regulated kinase1/2 pathway in RAW264.7 macrophages. The effects of ISL on LPS-induced NO and TNF-alpha production were reversed by the HO-1 inhibitor, tin protoporphyrin. CONCLUSIONS: ISL is an effective HO-1 inducer capable of inhibiting macrophage-derived inflammation.

Diabetes-induced colonic slow transit mediated by the up-regulation of PDGFRα+ cells/SK3 in streptozotocin-induced diabetic mice.

BACKGROUND: A major complication related to gastrointestinal (GI) symptoms in diabetic patients is chronic constipation. Constipation has serious negative impacts on quality of life; however, without a comprehensive understanding of the disease, currently available treatments cannot provide a cure. Platelet-derived growth factor receptor alpha-positive cells (PDGFRα+ cells), which form the SIP syncytium with interstitial cells of Cajal and smooth muscle cells, play important roles in GI motility. In the present study, the contributions of PDGFRα+ cells to diabetes-induced colonic slow transit were investigated in streptozotocin (STZ)-induced diabetic mice. METHODS: Western blotting, quantitative PCR, contractile experiments, and intracellular recording were used in the present study. KEY RESULTS: The results demonstrated that the colon length was increased in STZ-treated mice. The colonic transit of artificial fecal pellets in vitro was significantly delayed in STZ-treated mice. The mRNA and protein expression of PDGFRα, small-conductance Ca2+ -activated K channels (SK3), and P2Y1 receptors were increased in the colons of STZ-treated mice. In contractile experiments, the colonic smooth muscles were more sensitive to the SK3 agonist and antagonist (CyPPA and apamin) and the P2Y1 agonist and antagonist (MRS2365 and MRS2500) in STZ-treated mice. Intracellular recordings showed the responses of membrane potentials in colonic smooth muscle cells to CyPPA, apamin, MRS2365, and MRS2500 were more sensitive in STZ-treated mice. The electric field stimulation-induced P2Y1/SK3-dependent fast inhibitory junctional potentials (fIJPs) of colonic smooth muscles were more significantly hyperpolarized in STZ-treated mice. CONCLUSIONS AND INFERENCES: These results suggest that the purinergic neurotransmitters/P2Y1/SK3 signaling pathway is up-regulated in the diabetic colons, thereby mediating diabetes-induced colonic slow transit.

Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2.

The transition from interleukin-2 (IL-2)-dependent to IL-2-independent growth is considered one of the key steps in the transformation of human T-cell leukemia virus type-I (HTLV-I)-infected T cells. The expression of thioredoxin-binding protein-2 (TBP-2) is lost during the transition of HTLV-I-infected T-cell lines. Here, we analysed the mechanism of loss of TBP-2 expression and the role of TBP-2 in IL-2-dependent growth in the in vitro model to investigate multistep transformation of HTLV-I. CpGs in the TBP-2 gene are methylated in IL-2-independent but not in IL-2-dependent cells. Sequential treatment with 5-aza-2'-deoxycytidine and a histone deacetylase inhibitor augmented histone acetylation and TBP-2 expression, suggesting that loss of TBP-2 expression is due to DNA methylation and histone deacetylation. In IL-2-dependent cells, a basal level of TBP-2 expression was maintained by IL-2 associated with cellular growth, whereas TBP-2 expression was upregulated on deprivation of IL-2 associated with growth suppression. Overexpression of TBP-2 in IL-2-independent cells suppressed the growth and partially restored responsiveness to IL-2. Knockdown of TBP-2 caused the IL-2-dependent cells to show partial growth without IL-2. These results suggested that epigenetic silencing of the TBP-2 gene results in a loss of responsiveness to IL-2, contributing to uncontrolled IL-2-independent growth in HTLV-I-infected T-cell lines.

Classical reward conditioning in Drosophila melanogaster.

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.

Health benefits of traditional corn, beans, and pumpkin: in vitro studies for hyperglycemia and hypertension management.

Levels of obesity-linked non-insulin-dependent diabetes mellitus (NIDDM) and hypertension are highest among indigenous communities in North America. This is linked to changes in dietary pattern towards high calorie foods such as sugar, refined grain flour, and sweetened beverages. Therefore, a return to traditional dietary patterns may help to reduce these disease problems because of better balance of calories and beneficial nutrients. Further protective non-nutrient phenolic phytochemicals against NIDDM and hypertension are potentially high in these foods but less understood. In this study antidiabetic- and antihypertension-relevant potentials of phenolic phytochemicals were confirmed in select important traditional plant foods of indigenous communities such as pumpkin, beans, and maize using in vitro enzyme assays for -glucosidase, alpha-amylase, and angiotensin I-converting enzyme (ACE) inhibitory activities. In vitro inhibitory activities of these enzymes provide a strong biochemical rationale for further in vivo studies and dietary management strategy for NIDDM through the control of glucose absorption and reduction of associated hypertension. These enzyme inhibitory activities were further compared to total soluble phenolic content and antioxidant activity of the above-targeted plant foods. Pumpkin showed the best overall potential. Among the varieties of pumpkin extracts P5 (round orange) and P6 (spotted orange green) had high content of total phenolics and moderate antioxidant activity coupled to moderate to high alpha-glucosidase and ACE inhibitory activities. Therefore this phenolic antioxidant-enriched dietary strategy using specific traditional plant food combinations can generate a whole food profile that has the potential to reduce hyperglycemia-induced pathogenesis and also associated complications linked to cellular oxidation stress and hypertension.

Increased sagittal vertical axis is associated with less effective control of acute pain following vertebroplasty.

OBJECTIVES: Although vertebroplasty is very effective for relieving acute pain from an osteoporotic vertebral compression fracture, not all patients who undergo vertebroplasty receive the same degree of benefit from the procedure. In order to identify the ideal candidate for vertebroplasty, pre-operative prognostic demographic or clinico-radiological factors need to be identified. The objective of this study was to identify the pre-operative prognostic factors related to the effect of vertebroplasty on acute pain control using a cohort of surgically and non-surgically managed patients. PATIENTS AND METHODS: Patients with single-level acute osteoporotic vertebral compression fracture at thoracolumbar junction (T10 to L2) were followed. If the patients were not satisfied with acute pain reduction after a three-week conservative treatment, vertebroplasty was recommended. Pain assessment was carried out at the time of diagnosis, as well as three, four, six, and 12 weeks after the diagnosis. The effect of vertebroplasty, compared with conservative treatment, on back pain (visual analogue score, VAS) was analysed with the use of analysis-of-covariance models that adjusted for pre-operative VAS scores. RESULTS: A total of 342 patients finished the 12-week follow-up, and 120 patients underwent vertebroplasty (35.1%). The effect of vertebroplasty over conservative treatment was significant regardless of age, body mass index, medical comorbidity, previous fracture, pain duration, bone mineral density, degree of vertebral body compression, and canal encroachment. However, the effect of vertebroplasty was not significant at all time points in patients with increased sagittal vertical axis. CONCLUSIONS: For single-level acute osteoporotic vertebral compression fractures, the effect of vertebroplasty was less favourable in patients with increased sagittal vertical axis (> 5 cm) possible due to aggravation of kyphotic stress from walking imbalance.Cite this article: Y-C. Kim, D. H. Bok, H-G. Chang, S. W. Kim, M. S. Park, J. K. Oh, J. Kim, T-H. Kim. Increased sagittal vertical axis is associated with less effective control of acute pain following vertebroplasty. Bone Joint Res 2016;5:544-551. DOI: 10.1302/2046-3758.511.BJR-2016-0135.R1.

2,4,5-Trimethoxyldalbergiquinol promotes osteoblastic differentiation and mineralization via the BMP and Wnt/ß-catenin pathway.

Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of ß-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/ß-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis.

Exogenous H2 S enhances mice gastric smooth muscle tension through S-sulfhydration of KV 4.3, mediating the inhibition of the voltage-dependent potassium current.

BACKGROUND: Hydrogen sulfide (H2 S) has been shown to have an excitatory effect on gastric motility, but the underlying molecular mechanism is unclear. In this study, we aimed to investigate the possible targets of H2 S and determine how H2 S affects its target proteins during H2 S-induced contraction. METHODS: Patch-clamp and potentiometric fluorescence dye were utilized to measure the electrophysiological changes. The Biotin-switch assay was utilized to detect the protein S-sulfhydration. The isometric tension measurement was conducted too. KEY RESULTS: Exogenous H2 S enhanced the tonic contraction of gastric antral smooth muscle, and voltage-dependent potassium channel (KV ) blocker and Dithiothreitol (DTT, a reducing agent) abolished the excitatory effect of NaHS. Exogenous H2 S inhibited the fast inactivation component of the voltage-dependent potassium channel current (IKVfast ) in isolated gastric antral smooth muscle cells. H2 S inhibited the KV 4.3 current in H293 cells with heterologous expression of KV 4.3, but did not inhibit the KV 4.1 and KV 4.2 currents, which together contribute greatly to IKVfast . NaHS significantly decreased the membrane potential in cultured gastric smooth muscle cells, but the NaHS-induced depolarization was suppressed by knockdown of KV 4.3 and N-ethylamaleimide (NEM), a free thiol group blocker. In addition, NaHS sulfhydrated KV 4.3 in H293 cells and in gastric smooth muscle tissue. However, this S-sulfhydration was inhibited by NEM and DTT. Meanwhile the NaHS-induced inhibition of IKVfast and KV 4.3 was also blocked by NEM and DTT. CONCLUSIONS & INFERENCES: These results suggest that exogenous H2 S sulfhydrates KV 4.3 to decrease the membrane potential, thereby enhancing the basal tension of gastric antral smooth muscle.

Comparison of medial and lateral meniscal transplantation with regard to extrusion of the allograft, and its correlation with clinical outcome.

We compared extrusion of the allograft after medial and lateral meniscal allograft transplantation and examined the correlation between the extent of extrusion and the clinical outcome. A total of 73 lateral and 26 medial meniscus allografts were evaluated by MRI at a mean of 32 months (24 to 59) in 99 patients (67 men, 32 women) with a mean age of 35 years (21 to 52). The absolute values and the proportional widths of extruded menisci as a percentage were measured in coronal images that showed maximum extrusion. Functional assessments were performed using Lysholm scores. The mean extrusion was 4.7 mm (1.8 to 7.7) for lateral menisci and 2.9 mm (1.2 to 6.5) for medial menisci (p < 0.001), and the mean percentage extrusions were 52.0% (23.8% to 81.8%) and 31.2% (11.6% to 63.4%), respectively (p < 0.001). Mean Lysholm scores increased significantly from 49.0 (10 to 83) pre-operatively to 86.6 (33 to 99) at final follow-up for lateral menisci (p = 0.001) and from 50.9 (15 to 88) to 88.3 (32 to 100) for medial menisci (p < 0.001). The final mean Lysholm scores were similar in the two groups (p = 0.312). Furthermore, Lysholm scores were not found to be correlated with degree of extrusion (p = 0.242). Thus, transplanted lateral menisci extrude more significantly than transplanted medial menisci. However, the clinical outcome after meniscal transplantation was not found to be adversely affected by extrusion of the allograft.

Radical scavenging-linked antioxidant activity of ethanolic extracts of diverse types of extra virgin olive oils.

The present study evaluated the radical scavenging-linked antioxidant activity of hexane/80% ethanol extracts from several types of extra virgin olive oils (EVOOs) derived from varieties arbequina, hojiblanca, picual, their blends, and pure olive oil (POO). The antioxidant potential of the olive oil extracts was assessed by radical scavenging assays using DPPH (2, 2-diphenyl-1-picrylhydrazyl), ABTS (2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), and hydroxyl radical, as well as hydrogen peroxide and superoxide anion inhibitory activities. Electron donating ability (EDA) using DPPH assay of 80% ethanol extracts from EVOOs, except arbequina oil, was significantly higher than POO. EDA was markedly higher in blended and picual EVOOs than the extracts from arbequina and hojiblanca EVOOs (P < 0.05). Similarly, ABTS radical scavenging activity of the extracts from the EVOOs was in order of picual EVOO > blended EVOO > hojiblanca EVOO >or= POO >or= arbequina EVOO. Further, the superoxide anion scavenging activity of blended, picual, and arbequina EVOOs was significantly higher than that of hojiblanca EVOO and POO, which were barely detectable. Hydroxyl radical scavenging activity of arbequina and hojiblanca was higher than that of blended, picual EVOOs, and POO. In addition, hydrogen peroxide scavenging activity of the extracts from blended, arbequina, hojiblanca, picual EVOOs, and POO was 63.1 +/- 3.1%, 44.4 +/- 10.2%, 52.0 +/- 2.7%, 71.8 +/- 2.5%, and 35.7 +/- 10.0%, respectively. Our results indicate that ethanol extracts of several EVOOs contained higher radical scavenging and antioxidant activity than the POO. This antioxidant potential is partly due to the phenolic compounds present in different olive oil grade and is influenced by cultivar type.