NOVA1 promotes SMN2 exon 7 splicing by binding the UCAC motif and increases SMN protein expression.

Spinal muscular atrophy (SMA) is a rare hereditary neuromuscular disease with a high lethality rate in infants. Variants in the homologous genes survival of motor neuron (SMN)1 and SMN2 have been reported to be SMA pathogenic factors. Previous studies showed that a high inclusion rate of SMN2 exon 7 increased SMN expression, which in turn reduced the severity of SMA. The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues. Neuro-oncological ventral antigen (NOVA) is a splicing factor that is specifically and highly expressed in neurons. It plays a key role in nervous system development and in the induction of nervous system diseases. However, it remains unclear whether this splicing factor affects SMA. In this study, we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA (genotype smn-/-SMN22tg/0) and littermate controls (genotype smn+/-SMN22tg/0). We found that inclusion level of SMN2 exon 7 was high in the brain and spinal cord tissue, and that NOVA1 was also highly expressed in nervous system tissues. In addition, SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system. We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1-7, and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed. We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression of the SMN2 protein in the U87MG cell line, whereas the opposite was observed when NOVA1 was knocked down. Finally, point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7. Moreover, CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif. Collectively, these findings indicate that NOVA1 interacts with the UCAC motif in exon 7 of SMN2, thereby enhancing inclusion of exon 7 in SMN2, which in turn increases expression of the SMN protein.

The Role and Molecular Mechanism of Long Nocoding RNA-MEG3 in the Pathogenesis of Preeclampsia.

A growing body of evidence suggests that the dysregulation of long noncoding RNA is increasingly linked to many human diseases. Maternally expressed gene 3 ( MEG3) is one such gene thought to be affected. In the placenta of patients with preeclampsia, there is reduced expression of MEG3; however, its role and the mechanism involved are not clear. Therefore, we examined the expression of MEG3, epithelial-mesenchymal transition (EMT) markers (E-cadherin and N-cadherin), and TGF-ß/smad signaling pathway genes ( TGF-ß1, smad3, and smad7) in the placental tissues of 20 patients with preeclampsia and 20 healthy patients. We further observed the impact of MEG3 on the invasion and migration functions of human trophoblast cells and the effects on EMT and TGF-ß/smad signaling pathways in an Human trophoblast cell-8 (HTR-8)Vneo cell line. The expression of MEG3 was lower in tissues from patients with preeclampsia having an EMT decline, as well as a messenger RNA expression of smad7. The expression of TGF-ß1 and smad3 were higher in patients with preeclampsia. In HTR-8/SVneo cells with overexpressed MEG3, the invasion and migration functions were enhanced and accompanied by higher EMT and a significantly increased expression of smad7. Our data indicate that MEG3 is closely associated with the pathogenesis of preeclampsia and thus associated with changes in the EMT of placental trophoblast cells. These results indicate that MEG3 regulation of trophoblast cell EMT via the TGF-ß pathway inhibitor smad7 may be the molecular mechanism involved in the pathogenesis of preeclampsia.

Mineralocorticoid Receptor Deficiency in T Cells Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction Through Modulating T-Cell Activation.

Although antagonists of mineralocorticoid receptor (MR) have been widely used to treat heart failure, the underlying mechanisms are incompletely understood. Recent reports show that T cells play important roles in pathologic cardiac hypertrophy and heart failure. However, it is unclear whether and how MR functions in T cells under these pathologic conditions. We found that MR antagonist suppressed abdominal aortic constriction-induced cardiac hypertrophy and decreased the accumulation and activation of CD4+ and CD8+ T cells in mouse heart. T-cell MR knockout mice manifested suppressed cardiac hypertrophy, fibrosis, and dysfunction compared with littermate control mice after abdominal aortic constriction. T-cell MR knockout mice had less cardiac inflammatory response, which was illustrated by decreased accumulation of myeloid cells and reduced expression of inflammatory cytokines. Less amounts and activation of T cells were observed in the heart of T-cell MR knockout mice after abdominal aortic constriction. In vitro studies showed that both MR antagonism and deficiency repressed activation of T cells, whereas MR overexpression elevated activation of T cells. These results demonstrated that MR blockade in T cells protected against abdominal aortic constriction-induced cardiac hypertrophy and dysfunction. Mechanistically, MR directly regulated T-cell activation and modulated cardiac inflammation. Targeting MR in T cells specifically may be a feasible strategy for more effective treatment of pathologic cardiac hypertrophy and heart failure.

Novel heterozygous mutation c.4282G>T in the SCN5A gene in a family with Brugada syndrome.

Brugada syndrome (BrS) is a rare, inherited arrhythmia syndrome. The most well-known gene that is responsible for causing BrS is SCN5A, which encodes the human cardiac Na+ channel (Nav1.5) α subunit. To date, it has been reported that >100 mutations in SCN5A can cause BrS. In the present study, a novel BrS-associated Nav1.5 mutation, A1428S, was identified in a proband who was successfully resuscitated from an episode of sudden collapse during walking. This mutation was further confirmed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis, which showed that the PCR fragment containing the mutation A1428S could be cut by the restriction enzyme Nsi1, yielding two shorter DNA fragments of 329 and 159 bp, which were not present in family members homozygous for the wild-type (WT) allele. Furthermore, the electrophysiological properties were analyzed by patch clamp technique. Current density was decreased in the A1428S mutant compared that in the WT. However, neither the steady-state activation or inactivation, nor the recovery from inactivation exhibited changes between the A1428S mutant and the WT. In conclusion, the results of this study are consistent with the hypothesis that a reduction in Nav1.5 channel function is involved in the pathogenesis of BrS. The structural-functional study of the Nav1.5 channel enhances the present understanding the pathophysiological function of the channel.

Interleukin-17A contributes to the development of post-operative atrial fibrillation by regulating inflammation and fibrosis in rats with sterile pericarditis.

Post-operative atrial fibrillation (AF) remains a common cause of morbidity. Increasing evidence indicates that inflammation and atrial fibrosis contribute to the pathogenesis of this condition. Interleukin (IL)-17A, a potent pro-inflammatory cytokine, has been implicated in the development of a number of cardiovascular diseases. However, its role in post-operative AF remains unknown. In the present study, sterile pericarditis (SP) was induced in rats by the epicardial application of sterile talc. AF was induced by transesophageal burst pacing. Western blot analysis was applied to quantify the expression of IL-17A. Quantitative PCR was used to detect the mRNA expression of IL-17A, IL-6, IL-1ß, transforming growth factor-ß1 (TGF-ß1), collagen type 1 (Col-1), collagen type 3 (Col-3) and α-smooth muscle actin (α-SMA). Gelatin zymography and reverse gelatin zymography were used to quantify the levels of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Histological analyses were performed to determine the extent of tissue inflammation and fibrosis. The rats with SP presented with a shorter refractoriness, a higher incidence and duration of AF, an enhanced susceptibility to developing AF, increased mRNA levels of AF-related pro-inflammatory cytokines (IL-6, IL-1ß and TGF-ß1), as well as marked atrial inflammation and fibrosis. The atrial IL-17A levels were elevated and correlated with the probability of developing AF. Treatment with anti-IL-17A monoclonal antibody decreased the levels of atrial IL-17A, prolonged refraction and markedly suppressed the development of AF. Simultaneously, inflammation and fibrosis were alleviated, which was further demonstrated by a decreased expression of AF-related pro-inflammatory cytokines, a downregulation in fibrosis-related mRNA expression (Col-1, Col-3 and α-SMA) and by the decreased activity of MMP-2/9 and TIMPs. Thus, the findings of our study indicate that IL-17A may play a pathogenic role in post-operative AF by inducing inflammation and fibrosis in rats with SP.

Acacetin blocks kv1.3 channels and inhibits human T cell activation.

BACKGROUNDS/AIMS: Acacetin, a natural flavonoid compound, has been proven to exert anti-inflammatory and immunomodulatory effects. Kv1.3 channels, highly expressed in human T cells, are attractive therapeutic targets to treat inflammatory and immunological disorders. The present study was designed to characterize the inhibition of Kv1.3 channels by Acacetin in human T cells and examine its role in T cell activation. METHODS: Whole-cell patch-clamp was applied to record the Kv1.3 and KCa currents in human T cells; Western blot was used to detect Kv1.3 expression as well as NFAT1 and NF-κB activity; Fluo-4, CCK-8 and an ELISA kit were used to measure Ca(2+) influx, proliferation, and IL-2 secretion, respectively. RESULTS: Acacetin decreased the Kv1.3 current, accelerated the decay rate and negatively shifted the steady-state inactivation curves in a concentration-dependent manner. The IC50 values at +40 mV for peak and the current at end of pulse were 21.09 ± 2.75 and 3.63 ± 0.25 µmol/L, respectively. Treatment with Acacetin for 24 h significantly inhibited Kv1.3 protein expression. Additionally, paralleling Kv1.3 inhibition, Acacetin also inhibited Ca(2+) influx, the Ca(2+)-activated transcription factors NFAT1, NF-κB p65/p50 activity, and proliferation as well as IL-2 production. Small interfering RNA against Kv1.3 reduced the inhibitory effect of Acacetin on IL-2 secretion. CONCLUSIONS: Acacetin blocks the Kv1.3 channel and inhibits human T cell activation. This action most likely contributes to its immunomodulatory and anti-inflammatory actions.

Lipid levels in serum and cancerous tissues of colorectal cancer patients.

AIM: To investigate the correlations between lipid metabolism disorder and the occurrence and development of colorectal cancer by monitoring the alterations in lipid levels in cancerous tissue and serum in patients with colorectal cancer. METHODS: The levels of total and free cholesterol (TCH and FCH), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein- cholesterol (HDL-C), apolipoprotein A1 (ApoA-1) and ApoB in serum of 206 patients with colorectal cancer, 70 patients with benign colorectal disease and 300 healthy participants, and in the cancerous tissue and paracancerous tissue of 152 patients with colorectal cancer were measured with an Olympus 600 auto-biochemical analyzer. The obtained data were statistically analyzed. RESULTS: Serum FCH level was significantly higher (1.9 ± 0.4 mmol/L vs 1.3 ± 0.3 mmol/L, 1.9 ± 0.4 mmol/L vs 1.2 ± 0.4 mmol/L, P < 0.05), whereas serum levels of TCH, LDL-C, ApoA-I and ApoB were significantly lower in patients with colorectal cancer than in patients with benign colorectal disease and healthy controls. The levels of FCH and TG in cancerous tissue were significantly lower (14.5 ± 9.6 µmol/g vs 19.3 ± 13.9 µmol/g, P < 0.05; 16.3 ± 19.8 µmol/g vs 44.1 ± 38.1 µmol/g, P < 0.05), whereas HDL-C level was significantly higher (7.9 ± 4.5 µmol/g vs 5.7 ± 3.9 µmol/g, P < 0.01) in cancerous tissue than in paracancerous tissue. The levels of TCH and TG in serum and the levels of TCH and HDL-C in cancerous tissue in patients with colorectal cancer were significantly correlated with TNM stage. The levels of TCH and LDL-C in serum were significantly lower, whereas HDL-C level in cancerous tissue was significantly higher in patients with lymph node metastasis than in patients without lymph node metastasis. The levels of TCH, FCH, TG, HDL-C and LDL-C in cancerous tissue were not significantly different from those in paracancerous tissue. The serum levels of FCH and TG were significantly higher, whereas serum HDL-C levels were significantly lower in patients with rectum cancer than in patients with colon cancer. CONCLUSION: The disordered and abnormally altered levels of lipids in cancerous tissue and serum of patients with colorectal cancer may be correlated with the occurrence and development of colorectal cancer.

Enrichment of MTHFR 677 T in a Chinese long-lived cohort and its association with lipid modulation.

BACKGROUND: Variants in the Methylenetetrahydrofolate reductase (MTHFR) gene may result in a lowered catalytic activity and associate with subsequent elevated serum homocysteine (Hcy) concentration, abnormal DNA synthesis and methylation, cardiovascular risk, and unhealthy aging. Several investigations on the relationship of MTHFR C677T polymorphism with serum lipid profile and longevity have been conducted in some populations, but the findings remain mixed. Herein, we sought to look at the association between MTHFR C677T and lipid profile in a longevous cohort in Bama, a well-known home of longevity in China. METHODS: Genotyping of MTHFR C677T was undertaken in 516 long-lived inhabitants (aged 90 and older, long-lived group, LG) and 493 healthy controls (aged 60-75, non-long-lived group, non-LG) recruited from Bama area. Correlation between MTHFR genotypes and lipids was then evaluated. RESULTS: T allele and TT genotype were significantly more prevalent in LG (P=0.001 and 0.002, respectively), especially in females, than in non-LG. No difference in the tested lipid measures among MTHFR C677T genotypes was observed in LG, non-LG and total population (P>0.05 for all). However, female but not male T carriers exhibited higher TC and LDL-C levels than did T noncarriers in the total population and in LG after stratification by sex (P<0.05 for each). These differences did not however remain through further subdivision by hyperlipidemia and normolipidemia. CONCLUSION: The higher prevalence of MTHFR 677 T genotypes and its modest unfavorable impact on lipids in Bama long-lived individuals may imply an existence of other protective genotypes which require further determination.

Synergistic inhibition of breast cancer by co-delivery of VEGF siRNA and paclitaxel via vapreotide-modified core-shell nanoparticles.

A somatostatin analog, vapreotide (VAP), can be used as a ligand for targeting drug delivery based on its high affinity to somatostatin receptors (SSTRs), which is overexpressed in many tumor cells. RNA interference plays an important role on downregulation of vascular endothelial growth factor (VEGF), which is important for tumor growth, progression and metastasis. To improve tumor therapy efficacy, the vapreotide-modified core-shell type nanoparticles co-encapsulating VEGF targeted siRNA (siVEGF) and paclitaxel (PTX), termed as VAP-PLPC/siRNA NPs, were developed in this study. When targeted via somatostatin receptors to tumor cells, the VAP-PLPC/siRNA NPs could simultaneously delivery siVEGF and PTX into cells and achieve a synergistic inhibition of tumor growth. Interestingly, in vitro cell uptake and gene silencing experiments demonstrated that the targeted VAP-PLPC/siRNA NPs exhibited significant higher intracellular siRNA accumulation and VEGF downregulation in human breast cancer MCF-7 cells, compared to those of the non-targeted PEG-PLPC/siRNA NPs. More importantly, in vivo results further demonstrated that the targeted VAP-PLPC/siRNA NPs had significant stronger drug distribution in tumor tissues and tumor growth inhibition efficacy via receptor-mediated targeting delivery, accompany with an obvious inhibition of neovascularization induced by siVEGF silencing. These results suggested that the co-delivery of siRNA and paclitaxel via vapreotide-modified core-shell nanoparticles would be a promising approach for tumor targeted therapy.

Possible roles of insulin, IGF-1 and IGFBPs in initiation and progression of colorectal cancer.

AIM: To investigate the roles of serum insulin, insulin-like growth factor-1 (IGF-1), and insulin-like growth factor binding proteins (IGFBPs) in the initiation and progression of colorectal cancer. METHODS: We determined serum insulin, IGF-1 and IGFBPs levels in 615 colorectal cancer patients and 650 control healthy donors by enzyme-linked immunosorbent assay (ELISA). In the meantime, their body mass index (BMI) and waist-to-hip ratio (WHR) were measured. RESULTS: Serum levels of insulin and IGF-1 as well as IGF-1/IGFBP-3 ratio in pre-operation patients were significantly elevated, but the level of IGFBP-3 was significantly decreased compared with normal controls and post-operation patients (P < 0.05 and P < 0.001, respectively). There is no significant difference (P > 0.05) in the levels of insulin, IGF-1, IGFBP-1, IGFBP-3 and IGF-1/IGFBP-3 between the patients with and without hepatic as well as distal abdominal metastases. WHR and BMI of colon cancer patients were positively and significantly correlated with the levels of insulin and IGF-1/IGFBP-3. In contrast, WHR and BMI were negatively correlated with IGFBP-3 level. CONCLUSION: The elevation of insulin, IGF-1 as well as IGF-1/IGFBP-3 ratio and the reduction of IGFBP-3 may be related to the initiation of colorectal cancer, but they are not related to the progression and outcome of the disease.

Tumor markers for diagnosis, monitoring of recurrence and prognosis in patients with upper gastrointestinal tract cancer.

To evaluate the value of combined detection of serum CEA, CA19-9, CA24-2, AFP, CA72-4, SCC, TPA and TPS for the clinical diagnosis of upper gastrointestinal tract (GIT) cancer and to analyze the efficacy of these tumor markers (TMs) in evaluating curative effects and prognosis. A total of 573 patients with upper GIT cancer between January 2004 and December 2007 were enrolled in this study. Serum levels of CEA, CA19-9, CA24-2, AFP, CA72-4, SCC, TPA and TPS were examined preoperatively and every 3 months postoperatively by ELISA. The sensitivity of CEA, CA19-9, CA24-2, AFP, CA72-4, SCC, TPA and TPS were 26.8%, 36.2%, 42.9%, 2.84%, 25.4%, 34.6%, 34.2% and 30.9%, respectively. The combined detection of CEA+CA199+CA242+CA724 had higher sensitivity and specificity in gastric cancer (GC) and cardiac cancer, while CEA+CA199+CA242+SCC was the best combination of diagnosis for esophageal cancer (EC). Elevation of preoperative CEA, CA19-9 and CA24-2, SCC and CA72-4 was significantly associated with pathological types (p<0.05) and TNM staging (p<0.05). Correlation analysis showed that CA24-2 was significantly correlated with CA19-9 (r=0.810, p<0.001). The levels of CEA, CA19-9, CA24-2, CA72-4 and SCC decreased obviously 3 months after operations. When metastasis and recurrence occurred, the levels of TMs significantly increased. On multivariate analysis, high preoperative CA72-4, CA24-2 and SCC served as prognostic factors for cardiac carcinoma, GC and EC, respectively. combined detection of CEA+CA199+CA242+SCC proved to be the most economic and practical strategy in diagnosis of EC; CEA+CA199+CA242+CA724 proved to be a better evaluation indicator for cardiac cancer and GC. CEA and CA19-9, CA24-2, CA72-4 and SCC, examined postoperatively during follow-up, were useful to find early tumor recurrence and metastasis, and evaluate prognosis. AFP, TPA and TPS have no significant value in diagnosis of patients with upper GIT cancer.

Potent suppression of Kv1.3 potassium channel and IL-2 secretion by diphenyl phosphine oxide-1 in human T cells.

Diphenyl phosphine oxide-1 (DPO-1) is a potent Kv1.5 channel inhibitor that has therapeutic potential for the treatment of atrial fibrillation. Many other Kv1.5 channel blockers also potently inhibit the Kv1.3 channel, but whether DPO-1 blocks Kv1.3 channels has not been investigated. The Kv1.3 channel is highly expressed in activated T cells, which is considered a favorable target for immunomodulation. Accordingly, we hypothesized that DPO-1 may exert immunosuppressive and anti-inflammatory effects by inhibiting Kv1.3 channel activity. In this study, DPO-1 blocked Kv1.3 current in a voltage-dependent and concentration-dependent manner, with IC50 values of 2.58 µM in Jurkat cells and 3.11 µM in human peripheral blood T cells. DPO-1 also accelerated the inactivation rate and negatively shifted steady-state inactivation. Moreover, DPO-1 at 3 µM had no apparent effect on the Ca²âº activated potassium channel (K(Ca)) current in both Jurkat cells and human peripheral blood T cells. In Jurkat cells, pre-treatment with DPO-1 for 24 h decreased Kv1.3 current density, and protein expression by 48±6% and 60±9%, at 3 and 10 µM, respectively (both p<0.05). In addition, Ca²âº influx to Ca²âº-depleted cells was blunted and IL-2 production was also reduced in activated Jurkat cells. IL-2 secretion was also inhibited by the Kv1.3 inhibitors margatoxin and charybdotoxin. Our results demonstrate for the first time that that DPO-1, at clinically relevant concentrations, blocks Kv1.3 channels, decreases Kv1.3 channel expression and suppresses IL-2 secretion. Therefore, DPO-1 may be a useful treatment strategy for immunologic disorders.

18ß-Glycyrrhetinic acid potently inhibits Kv1.3 potassium channels and T cell activation in human Jurkat T cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice has been extensively used in traditional medicines for treatment of many diseases, including inflammations and immunological disorders. Recent studies have shown that the anti-inflammatory and immunomodulation activities of licorice have been attributed to its active component, glycyrretinic acid (GA). GA consists of two isoforms, 18α- and 18ß-. However, its mechanism remains poorly understood. AIM OF THE STUDY: We compared the effects of two isoforms on Kv1.3 channels in Jurkat T cells and further characterized the inhibition of Kv1.3 channels by 18ß-GA in CHO cells. In addition, we examined the effects of 18ß-GA on Kv1.3 gene expression, Ca(2+) influx, proliferation, as well as IL-2 production in Jurkat T cells. MATERIALS AND METHODS: Whole-cell patch-clamp technique was applied to record Kv1.3 currents in Jurkat T or CHO cells. Real-time PCR and Western blotting were used to detect gene expression. Fluo-4, CCK-8 kit and ELISA kit were used to measure Ca(2+) influx, proliferation, and IL-2 secretion in Jurkat T cells, respectively. RESULTS: Superfusion of 18ß-GA (10-100 µM) blocked Kv1.3 currents in Jurkat T cells, while 18α-GA at the same concentration had no effect. The 18ß-GA induced inhibition had a voltage- and concentration-dependent manner with an IC50 of 23.9±1.5 µM at +40 mV in CHO cells. Furthermore, 18ß-GA significantly inhibited Kv1.3 gene expression. In addition, paralleling Kv1.3 inhibition, 18ß-GA also inhibited Ca(2+) influx, proliferation as well as IL-2 production in Jurkat T cells. CONCLUSION: 18ß-GA blocks Kv1.3 channels, which probably involves its anti-inflammatory and immunomodulation effects.

[The dose-response relationship of chrysotile asbestos exposure and lung cancer in cohort study].

OBJECTIVE: To clarify the dose-response relationship between asbestos dust exposure and lung cancer incidence in chrysotile asbestos miners by fixed cohort study and to investigate the incidence rates of lung cancer in exposure to different concentrations of asbestos dust. METHODS: A retrospective cohort study was conducted in 1932 asbestos miners who registered from January 1, 1981 to December 31, 1988, had worked for at least 1 year, and had no obvious cardiopulmonary diseases; the cohort study began in July 2009 and covered a time span of 29 years (1981 - 2009). The personal information, occupational history, disease history, and health data of these miners were recorded, and the monitoring data on dust concentrations in the mine over the years were collected. The dose-response relationship between asbestos dust concentration and lung cancer incidence was established by the method of life table; a regression equation was fitted to predict the excess incidence rates of lung cancer under the conditions of different working years and dust concentrations. RESULTS: A significant dose-response relationship was observed between cumulative exposure (Ce) and cumulative probability (Px) of lung cancer incidence, and the smokers hada higher Px than nonsmokers. When Ce was less than 2000 mg/m(3)·each year, Px reached 6.58/10000; when Ce was not less than 2000 mg/m(3)·and less than 3000 mg/m(3)·each year, Px reached 91.72/10000; when Ce was more than 5000 mg/m(3)·each year, Px was as high as 141.02/10000. The three models were fitted to obtain the optimal regression equation: Px = -0.0004Ce(2) + 0.0052Ce - 0.0011 (r(2) = 0.9387). In the workshop of asbestos mine in this study, the average dust concentration was 85 times higher than the limit in 2009, so the excess incidence rate of lung cancer was 112.598/10000 if the miners worked under this condition for 40 years, according to the equation. CONCLUSION: There is a significant dose-response relationship between cumulative asbestos exposure and lung cancer incidence in chrysotile asbestos miners. The risk for lung cancer rises as asbestos exposure increases.

Blocking of the human ether-à-go-go-related gene channel by imatinib mesylate.

Imatinib mesylate (IM), a widely prescribed powerful tyrosine kinase inhibitor, has been associated with increased risk of heart failure and is known to induce cell apoptosis and death in isolated cardiomyocytes. In addition to acquired long QT syndrome, pharmacological inhibition of human ether-à-go-go-related gene (HERG) channel has been reported to involve in apoptosis. The present study was undertaken to characterize the biophysical properties of IM on HERG and the molecular determinants of HERG blockade using mutant channels (Y652A and F656A). Wild type (WT) and mutant HERG channels were expressed in HEK-293 cells and Xenopus oocytes and the currents (I(HERG)) were measured using patch-clamp and two-microelectrode voltage-clamp techniques. IM inhibited WT I(HERG) in a concentration-dependent manner with an IC(50) of 19.51±2.50 µmol/L and 44.76±1.54 µmol/L in HEK-293 cells and Xenopus oocytes, respectively. The IM-induced inhibition of WT I(HERG) followed a voltage- and time-dependent manner. The blockade was enhanced by further activation of currents, which were in accordance with an open-channel blockade. The V(1/2) for steady-state activation shifted from -15.48±1.21 to -26.66±2.98 mV (p<0.05, n=6). The inactivation kinetics and voltage dependence of steady-state inactivation of the WT HERG channel were not significantly altered by IM. Two S6 domain mutants, F652A and Y656A, attenuated IM-induced inhibition of WT I(HERG). Therefore, IM preferentially blocked the open HERG channel through F652 and Y656, providing a molecular mechanism for the cardiac side effects during the clinical administration of IM.

Hypoxia induces voltage-gated K+ (Kv) channel expression in pulmonary arterial smooth muscle cells through hypoxia-inducible factor-1 (HIF-1).

Hypoxia-inducible factor-1 (HIF-1) regulates the expression of hypoxia-inducible genes by binding erythropoietin (EPO) enhancer fragments. Of these genes, HIF-1 upregulates voltage-gated K+1.2 channels (Kv1.2) in rat PC12 cells. Whether HIF-1 regulates hypoxia-induced Kv channel expression in cultured pulmonary artery smooth muscle cells (PASMCs), however, has not been determined. In this study, we investigated the effects of hypoxia on the expression of Kv1.2 Kv1.5, Kv2.1, and Kv9.3 channels in PASMCs and examined the direct role of HIF-1 by transfecting either wild type or mutant EPO enhancer fragments. Our results showed that 18 h exposure to hypoxia significantly increased the expression of Kv1.2, Kv1.5, Kv2.1, and Kv9.3; and this hypoxia-induced upregulation was completely inhibited after transfection with the wild type but not mutant EPO enhancer fragment. These results indicate that HIF-1 regulates hypoxia-stimulated induction of Kv1.2 Kv1.5, Kv2.1, and Kv9.3 channels in cultured PASMCs.

High expression of p300 is linked to aggressive features and poor prognosis of nasopharyngeal carcinoma.

BACKGROUND: Increased expression of transcriptional coactivator p300 has been observed in a variety of human cancers. However, the expression status of p300 protein/mRNA in nasopharyngeal carcinoma (NPC) tissues and its clinicopathologic/prognostic implication are poorly understood. METHODS: In our study, mRNA and protein expression levels of p300 was explored by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting (WB) and immunohistochemistry (IHC) in nasopharyngeal mucosal and NPC tissues. The data were analyzed by receiver operating characteristic (ROC) curve analysis, spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model. RESULTS: Up-regulated expression of p300 mRNA/p300 protein was detected in NPC tissues by RT-PCR and WB, when compared to nasopharyngeal mucosal tissues. Based on ROC curve analysis, the cutoff score for p300 high expression was defined when more than 35% of the tumor cells were positively stained. High expression of p300 was observed in 127/209 (60.7%) of NPCs. In NPCs, high expression of p300 was positively associated with later T classification, later N classification, distant metastasis and later clinical stage (P < 0.05). In univariate survival analysis, overexpression of p300 was found to be an indicator of progression-free (P = 0.002) and overall survival (P = 0.001) in NPCs. More importantly, p300 expression was evaluated as an independent prognostic factor for NPC in multivariate analysis (P = 0.036). CONCLUSIONS: Our findings support that high expression of p300 protein might be important in conferring a more aggressive behavior, and is an independent molecular marker for shortened survival time of patients with NPC.

An effective and safe supplement for stem cells expansion ex vivo: cord blood serum.

Mesenchymal stem cells (MSCs) are potential and optimal stem cells in clinical cell therapy, and fetal bovine serum (FBS) is widely used for expansion of MSCs, despite the risks of infectious disease transmission and immunological reaction of the xenogenic origin. This study was designed to compare human four blood group cord blood serum (CBS) with FBS in culturing human placenta-derived mesenchymal stem cells (hPDMSCs), which were derived from four blood group donors. The expansion medium included: 10% FBS, 10% A-CBS, 10% B-CBS, 10% O-CBS, and 10% AB-CBS. Cumulative population doubling, generation time, fold expansion rates and differentiation capacity, cell cycle, and immunophenotype were also assessed. The results showed that fold expansion rate and cumulative population doubling of hPDMSCs significantly increased during long-term MSC expansion in CBS medium, but the generation time decreased compared with FBS. CBS might be an effective supplement for stem cells expand rapidly ex vivo. Cell cycle and differentiation assays showed that most of the hPDMSCs expanding in the presence of CBS were in stationary phase, which was the characteristic of stem cells, and they retained their ability to differentiate into chondrogenic and endothelial cells. By comparing these four blood groups of CBS, we found that there was no significant difference among different blood groups in culturing hPDMSCs, which were isolated from different blood group donors. So CBS may be an optimal replacement to avoid the risks of FBS application in expansion of stem cell for clinical cell therapy and tissue engineering.

Microsomal triglyceride transfer protein gene -493G/T polymorphism and its association with serum lipid levels in Bama Zhuang long-living families in China.

BACKGROUND: The -493G/T polymorphism in the microsomal triglyceride transfer protein (MTP) gene is associated with lower serum low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels and longevity in several populations, but the results are inconsistent in different racial/ethnic groups. The current study was to investigate the plausible association of MTP -493G/T polymorphism with serum lipid levels and longevity in Zhuang long-lived families residing in Bama area, a famous home of longevity in Guangxi, China. METHODS: The MTP -493G/T was genotyped by PCR-restriction fragment length polymorphism in 391 Bama Zhuang long-lived families (BLF, n = 1467, age 56.60 ± 29.43 years) and four control groups recruited from Bama and out-of-Bama area with or without a familial history of exceptional longevity: Bama non-long-lived families (BNLF, n = 586, age 44.81 ± 26.83 years), Bama non-Zhuang long-lived families (BNZLF, n = 444, age 52.09 ± 31.91 years), Pingguo long-lived families (PLF, n = 658, age 50.83 ± 30.30 years), and Pingguo non-long-lived families (PNLF, n = 539, age 38.74 ± 24.69 years). Correlation analyses between genotypes and serum lipid levels and longevity were then performed. RESULTS: No particularly favorable lipoprotein and clinical phenotypes were seen in BLF as compared to general families in the same area. Instead, the levels of total cholesterol (TC), TG, LDL-C, and the prevalence of dyslipidemia were significantly higher in the three Bama families as compared to the two non-Bama families (P < 0.01 for all). There were no differences in the allelic and genotypic frequencies among the tested cohorts (P > 0.05 for all), but the TT genotype tended to enrich in the three long-lived cohorts from both areas. In addition, the individuals harboring TT genotype exhibited lower LDL-C and TC levels in the overall populations and Bama populations with a region- and sex-specific pattern. Multiple linear regression analyses unraveled that LDL-C levels were correlated with genotypes in Bama combined population, BNLF, and the total population (P < 0.05 for each) but not in Pingguo populations; TC and HDL-C levels were correlated with genotypes in Bama combined population and BLF, respectively (P < 0.05 for each). CONCLUSIONS: MTP -493G/T polymorphism may play an important role in fashioning the serum lipid profiles of Bama populations, despite no direct association between MTP -493G/T and longevity was detected.