HNRNPA2B1-mediated m6A modification of lncRNA MEG3 facilitates tumorigenesis and metastasis of non-small cell lung cancer by regulating miR-21-5p/PTEN axis.

BACKGROUND: Accumulating data indicate that N6-methyladenosine (m6A) RNA methylation and lncRNA deregulation act crucial roles in cancer progression. Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) as an m6A "reader" has been reported to be an oncogene in multiple malignancies. We herein aimed to elucidate the role and underlying mechanism by which HNRNPA2B1-mediated m6A modification of lncRNAs contributes to non-small cell lung cancer (NSCLC). METHODS: The expression levels of HNRNPA2B1 and their association with the clinicopathological characteristics and prognosis in NSCLC were determined by RT-qPCR, Western blot, immunohistochemistry and TCGA dataset. Then, the role of HNRNPA2B1 in NSCLC cells was assessed by in vitro functional experiments and in vivo tumorigenesis and lung metastasis models. HNRNPA2B1-mediated m6A modification of lncRNAs was screened by m6A-lncRNA epi-transcriptomic microarray and verified by methylated RNA immunoprecipitation (Me-RIP). The lncRNA MEG3-specific binding with miR-21-5p was evaluated by luciferase gene report and RIP assays. The effects of HNRNPA2B1 and (or) lncRNA MEG3 on miR-21-5p/PTEN/PI3K/AKT signaling were examined by RT-qPCR and Western blot analyses. RESULTS: We found that upregulation of HNRNPA2B1 was associated with distant metastasis and poor survival, representing an independent prognostic factor in patients with NSCLC. Knockdown of HNRNPA2B1 impaired cell proliferation and metastasis in vitro and in vivo, whereas ectopic expression of HNRNPA2B1 possessed the opposite effects. Mechanical investigations revealed that lncRNA MEG3 was an m6A target of HNRNPA2B1 and inhibition of HNRNPA2B1 decreased MEG3 m6A levels but increased its mRNA levels. Furthermore, lncRNA MEG3 could act as a sponge of miR-21-5p to upregulate PTEN and inactivate PI3K/AKT signaling, leading to the suppression of cell proliferation and invasion. Low expression of lncRNA MEG3 or elevated expression of miR-21-5p indicated poor survival in patients with NSCLC. CONCLUSIONS: Our findings uncover that HNRNPA2B1-mediated m6A modification of lncRNA MEG3 promotes tumorigenesis and metastasis of NSCLC cells by regulating miR-21-5p/PTEN axis and may provide a therapeutic target for NSCLC.

EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner.

The regulation of cardiac differentiation is critical for maintaining normal cardiac development and function. The precise mechanisms whereby cardiac differentiation is regulated remain uncertain. Here, we have identified a GATA-4 target, EGF, which is essential for cardiogenesis and regulates cardiac differentiation in a dose- and time-dependent manner. Moreover, EGF demonstrates functional interaction with GATA-4 in inducing the cardiac differentiation of P19CL6 cells in a time- and dose-dependent manner. Biochemically, GATA-4 forms a complex with STAT3 to bind to the EGF promoter in response to EGF stimulation and cooperatively activate the EGF promoter. Functionally, the cooperation during EGF activation results in the subsequent activation of cyclin D1 expression, which partly accounts for the lack of additional induction of cardiac differentiation by the GATA-4/STAT3 complex. Thus, we propose a model in which the regulatory cascade of cardiac differentiation involves GATA-4, EGF, and cyclin D1.

[Research on Modeling Method for Chlorophyll Content Fine Measurement Based on Neural Network].

Aiming at SPAD values of living plant leaf chlorophyll content affected easily by the blade thickness, water content, etc, a fine retrieval method of chlorophyll content based on multiple parameters of neural network model is presented. The SPAD values and water index (WI) of leaves were obtained by the leaf transmittance under the irradiation of light central wavelength in 650 nm, 940 nm, 1450 nm respectively. Meanwhile, the corresponding blade thickness is got by micrometer and the chlorophyll content is measured by spectrophotometric method. To modeling samples, the single parameter model between SPAD values and chlorophyll content was built and the nonlinear model between WI, thickness, SPAD values and chlorophyll content was established based on BP neural network. The predicted value of chlorophyll content of test samples were calculated separately by two models, and the correlation and relative errors were analyzed between predicted values and actual values. 340 samples of three different plant leaves were tested by the method described above in experiment. The results showed that compared with single parameter model, the prediction accuracy of three different plant samples were improved in different degrees, the average absolute relative error of chlorophyll content of all pooled samples predicted by BP neural network model reduced from 7.55% to 5.22%. The fitting determination coefficient is increased from 0.83 to 0.93. The feasibility were verified in this paper that the prediction accuracy of living plant chlorophyll content can improved effectively using multiple parameter BP neural network model.

[Simulation of Atmospheric Nitrous Oxide Profiles Retrieval from AIRS Observations].

Nitrous Oxide is a very important greenhouse gases and ozone-depleting substances. Due to the limited observations, there are still many uncertainties to quantitatively describe the role of nitrous oxide played in both cases. We can retrieve the methane and carbon dioxide gas using thermal infrared satellite data AIRS, but it is rarely for the nitrous oxide retrieval. Therefore, this paper retrieves nitrous oxide profiles from the AIRS data with an Optimal Estimate Method for the first time in China. The issue of the a priori and channels election is discussed. Comparison of the retrieved AIRS profiles with HIPPO profiles show the retrieved profiles are in good agreement with the smoothed HIPPO profiles, and a notable improvement in this algorithm than the eigen vector regression algorithm. For pressures between 300 and 900 hPa, we got the most accurate profiles and the relative error is only 0.1%, which is consistent with the jacobian peaks of the selected channels.

Mitochondria-localizing triphenylphosphine-8-hydroxyquinoline Ru complexes induce ferroptosis and their antitumor evaluation.

Ruthenium complexes are one of the most promising anticancer drugs and ferroptosis is a novel form of regulated cell death, the study on the effect of Ru complexes on ferroptosis is helpful to find more effective antitumor drugs. Here, the synthesis and characterization of two Ru complexes containing 8-hydroxylquinoline and triphenylphosphine as ligands, [Ru(L1) (PPh3)2Cl2] (Ru-1), [Ru(L2) (PPh3)2Cl2] (Ru-2), were reported. Complexes Ru-1 âˆ¼ Ru-2 showed good anticancer activity in Hep-G2 cells. Researches indicated that complexes Ru-1 âˆ¼ Ru-2 could be enriched and appear as red fluorescence in the mitochondria, arouse dysfunction of mitochondria, induce the accumulation of reactive oxygen species (ROS) and lipid peroxidation (LPO), while the morphology of nuclei and cell apoptosis had no significant change. Further experiments proved that GPX4 and Ferritin were down-regulated, which eventually triggered ferroptosis in Hep-G2 cells. Remarkably, Ru-1 showed high inhibitory activity against xenograft tumor growth in vivo (TGIR = 49%). This study shows that the complex Ru-1 could act as a novel drug candidate by triggering cell ferroptosis.

Maternal and neonatal blood vitamin D status and neurodevelopment at 24 months of age: a prospective birth cohort study.

BACKGROUND: This study aimed to explore the relationship of 25-hydroxyvitamin D [25(OH)D] in three trimesters and at birth with neurodevelopment at 24 months of age. METHODS: From 2013 to 2016, pregnant women from the Shanghai Birth Cohort in China were recruited for the study. Altogether, 649 mother-infant pairs were included. Serum 25(OH)D was measured with mass spectrometry in three trimesters, and cord blood was divided into deficiency (< 20 and < 12 ng/mL, respectively), insufficiency (20-30 and 12-20 ng/mL, respectively), and sufficiency (≥ 30 and ≥ 20 ng/mL, respectively). Bayley-III scale was used to assess cognitive, language, motor, social-emotional, and adaptive behavior development at 24 months of age. The Bayley-III scores were grouped into quartiles, and scores within the lowest quartile were defined as suboptimal development. RESULTS: After adjusting for confounding factors, cord blood 25(OH)D in the sufficient group was positively correlated with cognitive [ß = 11.43, 95% confidence interval (CI) = 5.65-17.22], language (ß = 6.01, 95% CI = 1.67-10.3), and motor scores (ß = 6.43, 95% CI = 1.73-11.1); cord blood 25(OH)D in the insufficient group was also positively correlated with cognitive scores (ß = 9.42, 95% CI = 3.74-15.11). Additionally, sufficient vitamin D status in the four periods and persistent 25(OH)D ≥ 30 ng/mL throughout pregnancy were associated with a lower risk of suboptimal cognitive development in adjusted models, although the effects were attenuated after applying the false discovery rate adjustment. CONCLUSIONS: Cord blood 25(OH)D ≥ 12 ng/mL has a significant positive association with cognitive, language, and motor development at 24 months of age. Sufficient vitamin D status in pregnancy might be a protective factor for suboptimal neurocognition development at 24 months of age.

'Internet+' comprehensive nursing training course in the post-epidemic era-an exploration of the mixed teaching mode: a randomized trial.

Objective: To explore the effect of the application of the 'Internet+' nursing teaching mode on the comprehensive teaching 'Fundamentals of Nursing'. Trial design: Parallel design and convenient sampling were used to select vocational nursing students from the Nursing College of Capital Medical University. Methods: Selected students were randomly divided into two groups. The control group consisted of 30 students in Grade 2020 higher vocational nursing education (traditional teaching mode). The observation group consisted of 30 students in Grade 2021 higher vocational nursing education (Internet+ mixed teaching mode). Training assessment results, automatic learning ability, professional identity, and satisfaction were compared between the two groups. Results: Compared with the control group, the students in the observation group scored higher in the following operation practices: venous blood sampling, intradermal injection, cardiopulmonary resuscitation (CPR), sputum aspiration, and putting on and taking off robes (84.01 ± 0.87 vs. 92.14 ± 1.23; 91.41 ± 0.82 vs. 96.86 ± 0.27; 87.56 ± 0.31 vs. 93.91 ± 2.79; 88.11 ± 0.51 vs. 93.75 ± 0.29; and 82.29 ± 0.29 vs. 90.96 ± 0.34, respectively, with p < 0.05 for all scores). The total scores for autonomous learning ability and subjective satisfaction were also higher in the observation group compared with the control group (82.98 ± 4.72 vs. 93.17 ± 5.01 and 96.67% vs. 90.00%, respectively, with p < 0.05 for all scores). Conclusion: In the post-epidemic era, the 'Internet+ hybrid teaching mode' was applied to comprehensive nursing teaching. This changed the traditional education mode, which focuses only on professional knowledge. The 'Internet+' teaching mode results showed that the professional, ideological, and political courses exhibited the same value guidance, which improved students' independent learning ability, practical operation ability, professional identity, and satisfaction.

[Identification of a mosaic mutation of NF1 gene in a sporadic case of neurofibromatosis type 1].

OBJECTIVE: To detect NF1 gene mutation in a patient with neurofibromatosis type 1. METHODS: Five fragments encompassing the entire coding sequence of the NF1 gene were amplified with reverse transcription PCR. PCR products were directly sequenced. Suspected mutations were verified by sequencing of DNA amplified by PCR using genomic DNA as template. Corresponding exon of family members was also sequenced. Furthermore, the PCR products were inserted into a pGEM-T cloning vector to quantify cells carrying the mutation in different samples derived from the three embryonic layers. RESULTS: The proband's clinical manifestation was consistent with neurofibromatosis type 1. Sequence analysis has identified a novel heterozygous mutation c.7911 C to T (p.Q2510X) in exon 51 of the NF1 gene in the proband. The same mutation was also detected in peripheral blood cells, uroepithelial cells and oral mucosal cells of the proband, though the signals of uroepithelial cells were significantly weaker. By T cloning-sequencing, recombinants carrying the NF1 gene mutation respectively accounted for 42%, 36% and 12% of all peripheral blood cells, oral mucosal cells and uroepithelial cells . CONCLUSION: It is likely that a mutation of NF1 gene has occurred in early embryogenesis of the proband, which in turn has led to generalized mosaicism of neurofibromatosis type 1.

[The monitoring of haze from HJ-1].

With the urbanizing in China, haze days occur frequently, which have largely impacted air quality. In the present paper, based on haze physical properties, haze optical properties were calculated by Mie theory and apparent reflectance of haze in Environment Satellite 1 (HJ-1) channels was simulated by Radiative Transfer (RT) 3. Simulated results show that it is reasonable to extract the haze from apparent reflectance in the first and second channels. By Deep Blue algorithm, Haze Optical Depth (HOD) was retrieved from HJ-1 data supported by reflectance database from MODIS product. From HJ-1 data in 2009 over Beijing area, the haze days were monitored and validated by AERONET/PHOTONS Beijing site. The validation shows that the correlation coefficient of HOD is greater than 0.9, but HOD from HJ-1 is greater than that from ground-based measurements. The discussions show that the error from reflectance database is less than 0.1 and radiance resolution of HJ-1 needs to advance for haze monitoring.

Correlation between contrast-enhanced cone-beam breast computed tomography features and prognostic staging in breast cancer.

OBJECTIVE: To evaluate whether contrast-enhanced cone-beam breast CT (CE-CBBCT) features can risk-stratify prognostic stage in breast cancer. METHODS: Overall, 168 biopsy-proven breast cancer patients were analysed: 115 patients in the training set underwent scanning using v. 1.5 CE-CBBCT between August 2019 and December 2019, whereas 53 patients in the test set underwent scanning using v. 1.0 CE-CBBCT between May 2012 and August 2014. All patients were restaged according to the American Joint Committee on Cancer eighth edition prognostic staging system. Following the combination of CE-CBBCT imaging parameters and clinicopathological factors, predictors that were correlated with stratification of prognostic stage via logistic regression were analysed. Predictive performance was assessed according to the area under the receiver operating characteristic curve (AUC). Goodness-of-fit of the models was assessed using the Hosmer-Lemeshow test. RESULTS: As regards differentiation between prognostic stage (PS) I and II/III, increased tumour-to-breast volume ratio (TBR), rim enhancement pattern, and the presence of penetrating vessels were significant predictors for PS II/III disease (p < 0.05). The AUCs in the training and test sets were 0.967 [95% confidence interval (CI) 0.938-0.996; p < 0.001] and 0.896 (95% CI, 0.809-0.983; p = 0.001), respectively. Two features were selected in the training set of PS II vs III, including tumour volume [odds ratio (OR)=1.817, p = 0.019] and calcification (OR = 4.600, p = 0.040), achieving an AUC of 0.790 (95% CI, 0.636-0.944, p = 0.001). However, there was no significant difference in the test set of PS II vs III (P＞0.05). CONCLUSION: CE-CBBCT imaging biomarkers may provide a large amount of anatomical and radiobiological information for the pre-operative distinction of prognostic stage. ADVANCES IN KNOWLEDGE: CE-CBBCT features have distinctive promise for stratification of prognostic stage in breast cancer.

Requirement of the NF-κB pathway for induction of Wnt-5A by interleukin-1ß in condylar chondrocytes of the temporomandibular joint: functional crosstalk between the Wnt-5A and NF-κB signaling pathways.

OBJECTIVE: We have previously reported that interleukin-1ß (IL-1ß) up-regulates the expression of Wnt-5A and the activation of Wnt-5A signaling induces matrix metalloproteinase (MMP) through the c-Jun N-terminal kinase pathway in condylar chondrocytes (CCs) of the temporomandibular joint (TMJ). These results suggest that Wnt-5A could play an essential role in IL-1ß-mediated cartilage destruction. The objective of this study was to investigate the molecular mechanism underlying IL-1ß-induced up-regulation of Wnt-5A in TMJ CCs. METHODS: Primary CCs, limb chondrocytes (LCs) and SW1353 human chondrosarcoma cells were treated with IL-1ß in the presence or absent of BAY 11-7082 (an inhibitor of IκBα-phosphorylation). Then, expression of Wnt-5A was estimated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytofluorescence. Transient transfection of p65 expression vector and chromatin immunoprecipitation (ChIP) assay was performed to define the effect of p65 on Wnt-5A expression. RESULTS: IL-1ß up-regulated Wnt-5A expression at both the RNA and protein levels in articular chondrocytes. The inhibitor of IκBα-phosphorylation, BAY 11-7082, blocked the induction of Wnt-5A by IL-1ß in a dose-dependent manner. Moreover, experiments with overexpression of p65 and ChIP established that induction of Wnt-5A by IL-1ß is mediated through the NF-κB pathway, especially the p65 subunit. CONCLUSION: These results clarify the molecular mechanism underlying up-regulation of Wnt-5A by IL-1ß in chondrocytes, suggesting an important functional crosstalk between Wnt-5A and NF-κB signaling pathways. This finding provides new insights into the involvement of Wnt signaling in the cartilage destruction caused by arthritis.

Knockout fth1b affects early mineralization of zebrafish pharyngeal teeth.

OBJECTIVES: A study was conducted to explore the expression pattern and function of ferritin heavy polypeptide gene (fth1b) in zebrafish pharyngeal teeth development and lay the foundation for subsequent research on teeth development and mineralization. METHODS: The zebrafish embryos were harvested at 56, 72, 96, and 120 h after fertilization. The expression of fth1b in zebrafish pharyngeal teeth development was detected by whole embryo in situ hybridization and compared with the known pharyngeal teeth marker dlx2b. The specific knockout of fth1b gene was performed using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology. The development of zebrafish pharyngeal teeth was detected in the fth1b-/- mutant. RESULTS: The expression pattern of fth1b gene was very similar to that of the known zebrafish pharyngeal teeth marker dlx2b and was specifically expressed in the zebrafish pharyngeal teeth during development. After the specific knockout of the gene fth1b, the earliest gene that can be detect in zebrafish pharyngeal teeth-pitx2 was expressed normally during early development. The dlx2b expression was not significantly different from that of wild type zebrafish, but the mineralization of pharyngeal teeth in the mutant was weaker than that of wild type zebrafish. CONCLUSIONS: The gene fth1b is specifically expressed in zebrafish pharyngeal teeth and acts on their early mineralization.

The Pharmacokinetic Effect of Itraconazole and Voriconazole on Ripretinib in Beagle Dogs by UPLC-MS/MS Technique.

BACKGROUND: A new UPLC-MS/MS technique for the determination of ripretinib in beagle dog plasma was developed, and the pharmacokinetic effects of voriconazole and itraconazole on ripretinib in beagle dogs were studied. METHODS: After extraction with ethyl acetate under alkaline conditions, ripretinib was detected using avapritinib as the internal standard (IS). The mobile phases were 0.1% formic acid-acetonitrile. The scanning method was multi-reaction monitoring using ESI+ source, and the ion pairs for ripretinib and IS were m/z 509.93→416.85 and 499.1→482.09, respectively. This animal experiment adopted a three period self-control experimental design. In the first period, ripretinib was orally administered to six beagle dogs at a dose of 5 mg/kg. In the second period, the same six beagle dogs were orally given itraconazole at a dose of 7 mg/kg, after 30 min, ripretinib was orally given. In the third period, voriconazole at a dose of 7 mg/kg was given orally, and then ripretinib was orally given. At different time points, the blood samples were collected. The concentration of ripretinib was detected, and the pharmacokinetic parameters of ripretinib were calculated. RESULTS: Ripretinib had a good linear relationship in the range of 1-1000 ng/mL. The precision, accuracy, recovery, matrix effect and stability met the requirements of the guiding principles. After erdafitinib combined with itraconazole, the Cmax and AUC0→t of ripretinib increased by 38.35% and 36.36%, respectively, and the t1/2 was prolonged to 7.53 h. After ripretinib combined with voriconazole, the Cmax and AUC0→t of ripretinib increased by 37.44% and 25.52%, respectively, and the t1/2 was prolonged to 7.33 h. CONCLUSION: A new and reliable UPLC-MS/MS technique was fully optimized and developed to detect the concentration of ripretinib in beagle dog plasma. Itraconazole and voriconazole could inhibit the metabolism of ripretinib in beagle dogs and increase the plasma exposure of ripretinib.

Establishment and Verification of UPLC-MS/MS Technique for Pharmacokinetic Drug-Drug Interactions of Selinexor with Posaconazole in Rats.

BACKGROUND: A method for the determination of selinexor by UPLC-MS/MS was established to study the effect of posaconazole on the pharmacokinetics of selinexor in rats. METHODS: The experiment rats were divided into group A (0.5% CMC-Na) and group B (posaconazole, 20 mg/kg), 6 rats in each group. 30 minutes after administration of 0.5% CMC-Na or posaconazole, all the rats were given selinexor (8 mg/kg), and plasma samples were collected. The plasma samples underwent acetonitrile protein precipitation, and were separated by UPLC on an Acquity UPLC BEH C18 column with gradient elution. Acetonitrile and 0.1% formic acid were used as the mobile phases. The analyte detection was used a Xevo TQ-S triple quadrupole tandem mass spectrometer and multiple reaction monitoring (MRM) for analyte monitoring. We use acetonitrile for protein precipitation. RESULTS: Selinexor had good linearity (1.0-1000 ng/mL, r2 =0.996 2), and the accuracy and precision, recovery rate and matrix effects(ME) were also met the FDA approval guidelines. Compared with group A, the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group B increased by 60.33%, 48.28% and 48.27%, and Tmax increased by 53.92%, CLz/F reduced by 32.08%. CONCLUSION: This bioanalysis method had been applied to the study of drug interactions in rats. It was found that posaconazole significantly increased the concentration of selinexor in rats. Therefore, when selinexor and posaconazole are combined, we should pay attention to the possible drug-drug interactions to reduce adverse reactions.

[Dual-role regulations of canonical Wnt/beta-catenin signaling pathway].

In recent years, Wnt/beta-catenin signaling has been identified as a key player in embryogenesis and human diseases. Canonical Wnt signaling pathway is controlled by a variety of classic molecules like Wnt, beta-catenin, Axin, APC, GSK-3beta and CK1, which interact and coordinate to regulate the expressions of cell signaling molecules. The latest evidences suggest that some components of the Wnt/beta-catenin signaling, like APC, GSK-3beta, CK1, Dkk2 and WISE, play dual roles different from what they have been thought previously. Here we reviewed some recent discoveries on the canonical Wnt/beta-catenin signaling pathway to provide some new ideas and principles for signaling transduction studies.

Aloe-emodin induces hepatotoxicity by the inhibition of multidrug resistance protein 2.

BACKGROUND: Aloe-emodin (AE) is among the primary bioactive anthraquinones present in traditional Chinese medicinal plants such as Rheum palmatum L. Multidrug resistance protein 2 (ABCC2/ MRP2) is an important efflux transporter of substances associated with cellular oxidative stress. However, the effects of traditional Chinese medicine on this protein remain unclear. PURPOSE: The aim of this research is to study the role of ABCC2 in AE-induced hepatotoxicity. METHODS: The expression of ABCC2 protein and mRNA levels were analyzed by Western-Blotting and qRT-PCR, respectively. The intracellular oxidative stress caused by AE was evaluated by quantifying the levels of intracellular reactive oxygen species, malondialdehyde, glutathione reduced and oxidized glutathione. The levels of adenosine triphosphate, mitochondrial membrane potential and mitochondrial DNA were explored to evaluate the effects of AE on mitochondrial function. The effects of AE on cell apoptosis and cell cycle were detected by flow cytometry. To further clarify the key role of ABCC2 in AE induced cytotoxicity, we used pCI-neo-ABCC2 plasmid to over express ABCC2 protein, and small interfering RNA was used to knockdown ABCC2 in HepG2 cells. Additionally, we investigated the impact of AE on ABCC2 degradation pathway and the hepatotoxic effects of AE in mice. RESULTS: AE was found to inhibit ABCC2 transport activity, downregulate ABCC2 expression and altered intracellular redox balance. Induction of oxidative stress resulted in depletion of intracellular glutathione reduced, mitochondria dysfunction and activation of apoptosis. ABCC2 overexpression significantly reduced AE-induced intracellular oxidative stress and cell death, which was enhanced by ABCC2 knockdown. Furthermore, AE was observed to promote ABCC2 degradation through induction of autophagy and hepatotoxicity was induced in mice by promoting ABCC2 degradation. CONCLUSIONS: The inhibition of ABCC2 is a novel effect of AE that triggers oxidative stress and apoptosis. These findings are helpful in understanding the toxicological effects of AE-containing medicinal plants.

beta-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes.

beta-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of beta-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which beta-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of beta-catenin onto the Anf promoter. Thus, we document a direct positive role of beta-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for beta-catenin/TCF/LEF1.

Protolimonoids from Melia toosendan.

Toosendanone A (1), a new euphane (tirucallane)-type triterpene bearing a five-membered ring in the side chain and the first cyclopentanyl protolimonoid, was isolated from the bark of Melia toosendan, along with two new tirucallanes, toosendanic acids A (2) and B (3). The structure and absolute configuration of compound 1 was elucidated by spectroscopic data interpretation and X-ray diffraction analysis. Compounds 1-3 were evaluated for cytotoxicity against a small panel of cancer cell lines.

[Redox factor 1 increases proliferation of neonatal rat cardiac fibroblasts].

OBJECTIVE: To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying mechanisms. METHODS: Neonatal rat cardiac fibroblasts were transfected with the adenoviral vector containing rat wild type Ref1 (Ad-Ref1) or mutated Ref1 (Ad-mutRef1). The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REF1 protein. MTT was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Ref1, collagen I (Col I) and collagen III (Col III) were determined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay (EMSA) was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium (25 mmol/L) was applied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expression and translocation of REF1 were examined. RESULTS: MTT analysis showed that Ad-Ref1 promoted the relative viability of cardiac fibroblasts (0.671+/-0.044 vs control 0.364+/-0.007, n=6, P<0.01). The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Ref1 transfected cells (16.8%+/-0.62% vs control 9.04%+/-0.43%, n=3, P<0.05), as demonstrated by flow cytometry analysis. The expressions of Col I and Col III at mRNA level were increased when cells transfected with Ad-Ref1, while Ad-mutRef1 did not show such effects. Compared with the redox-deficient mutant Ad-mutRef1 (C65/93A), EMSA results demonstrated that Ad-Ref1 resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose (25 mmol/L) medium resulted in an increase in AP1 DNA binding activity, which was similar as seen in Ad-Ref1 transfected cells. There was also an increased accumulation of nuclear REF1 protein when cells were cultured in high glucose medium, although the expressions of REF1 at both mRNA and protein levels were not affected. CONCLUSION: REF1 can increase proliferation and collagen synthesis of cardiac fibroblasts, which may be related to its ability to up-regulate AP1 DNA binding.

Carboxyl terminus of Nkx2.5 impairs its interaction with p300.

The transcription factor Nkx2.5 plays critical roles in controlling cardiac-specific gene expression. Previous reports demonstrated that Nkx2.5 is only a modest transactivator due to the auto-inhibitory effect of its C-terminal domain. Deletion of the C-terminal domain, mimicking conformational change, evokes vigorous transactivation activity. Here, we show that a C-terminal defective mutant of Nkx2.5 improves the occupation of p300 at the ANF promoter compared with full-length Nkx2.5, leading to hyperacetylation of histone H4. We reveal that p300 is a cofactor of Nkx2.5, markedly potentiating Nkx2.5-dependent transactivation, whereas E1A antigen impairs Nkx2.5 activity. Furthermore, p300 can acetylate Nkx2.5 and display an acetyltransferase-independent mechanism to coactivate Nkx2.5. Physical interaction between the N-terminal activation domain of Nkx2.5 and the C/H3 domain of p300 are identified by GST pull-down assay. Point mutants of the N-terminal modify the transcriptional activity of Nkx2.5 and interaction with p300. Deletion of the C-terminal domain greatly facilitates p300 binding and improves the susceptibility of Nkx2.5 to histone deacetylase inhibitor. These results establish that p300 acts as an Nkx2.5 cofactor and facilitates increased Nkx2.5 activity by relieving the conformational impediment of its inhibitory C-terminal domain.