The THO/TREX Complex Component RAE2/TEX1 Is Involved in the Regulation of Aluminum Resistance and Low Phosphate Response in Arabidopsis.

The C2H2-type zinc finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) plays a critical role in aluminum (Al) resistance and low phosphate (Pi) response mainly through promoting the expression of the malate transporter-encoding gene ARABIDOPSIS THALIANA ALUMINUM ACTIVATED MALATE TRANSPORTER 1 (AtALMT1). We previously showed that REGULATION OF ATALMT1 EXPRESSION 3 (RAE3/HPR1), a core component of the THO/TREX complex, is involved in the regulation of nucleocytoplasmic STOP1 mRNA export to modulate Al resistance and low Pi response. Here, we report that RAE2/TEX1, another core component of the THO complex, is also involved in the regulation of Al resistance and low Pi response. Mutation of RAE2 reduced the expression of STOP1-downstream genes, including AtALMT1. rae2 was less sensitive to Al than rae3, which was consistent with less amount of malate secreted from rae3 roots than from rae2 roots. Nevertheless, low Pi response was impaired more in rae2 than in rae3, suggesting that RAE2 also regulates AtALMT1-independent pathway to modulate low Pi response. Furthermore, unlike RAE3 that regulates STOP1 mRNA export, mutating RAE2 did not affect STOP1 mRNA accumulation in the nucleus, although STOP1 protein level was reduced in rae2. Introduction of rae1 mutation into rae2 mutant background could partially recover the deficient phenotypes of rae2. Together, our results demonstrate that RAE2 and RAE3 play overlapping but distinct roles in the modulation of Al resistance and low Pi response.

Prognostic significance of contactin 3 expression and associated genes in glioblastoma multiforme.

Contactin 3 (CNTN3) is a member of the contactin family that is primarily expressed in the nervous system. However, to the best of our knowledge, expression of contactin and its role in the development and progression of brain tumours has not been studied. Although glioblastoma multiforme (GBM) is the most common malignant brain tumour, advances in therapeutic options for patients with GBM have been modest due to an incomplete understanding of the molecular mechanisms underlying development and progression. The aim of the present study was to examine the correlation between CNTN3 and its associated genes and the clinical outcome in patients with GBM. CNTN3 and the expression levels of associated genes were analysed in GBM datasets obtained from the SAGE Anatomical viewer website, Gene Expression Omnibus, Oncomine and The Cancer Genome Atlas. CNTN3 was significantly downregulated in patients with GBM. Subsequently, the expression of CNTN3 was further validated using immunohistochemistry in a cohort of GBM specimens. The immunohistochemistry results were consistent with the in silico analyses. Kaplan-Meier analysis indicated that patients with lower expression levels of CNTN3 had a significantly shorter overall survival (OS) time compared with patients with higher levels of CNTN3 expression. Univariate and multivariate Cox regression analyses demonstrated that CNTN3 expression was an independent prognostic indicator in patients with GBM. Furthermore, gene set enrichment analysis revealed that CNTN3 was associated with the receptor tyrosine-protein kinase (ErbB) signalling pathway. In the ErbB signalling pathway, epidermal growth factor receptor (EGFR) was negatively correlated with CNTN3. Taken together, these data suggest that lower expression levels of CNTN3 may be an independent biomarker that predicts poor OS time in patients with GBM, and that EGFR expression in the ErbB pathway may be associated with CNTN3 expression.

Randomized controlled trial: moxibustion and acupuncture for the treatment of Crohn's disease.

AIM: To evaluate the clinical efficacy and safety of acupuncture and moxibustion for the treatment of active Crohn's disease (CD). METHODS: Ninety-two patients were equally and randomly divided into the treatment group and received herb-partitioned moxibustion combined with acupuncture, and the control group received wheat bran-partitioned moxibustion combined with superficial acupuncture. The patients received three treatment sessions per week for 12 wk and were followed up for 24 wk. The main outcome was evaluated using the CD Activity Index (CDAI) score, and the secondary outcomes were evaluated using laboratory indicators such as hemoglobin (HGB), C-reactive protein (CRP), erythrocyte sedimentation rate, quality-of-life, endoscopic ratings, and intestinal histology scores. RESULTS: The CDAI scores of both the treatment and control groups were significantly reduced after treatment compared with those measured before treatment. However, the degree of improvement in the treatment group was significantly greater than that of the control group. The improvement in symptoms in patients of the treatment group was sustained at follow-up, whereas that of the control group was not. The overall efficacy of the treatment was significantly greater than that of the control. Both groups demonstrated significant improvements in quality-of-life ratings after treatment, but the improvement was significantly greater in the treatment group than in the control group. In addition, the patients in the treatment group showed significantly increased HGB and significantly decreased CRP levels and histopathological scores at the end of treatment, whereas the control group did not exhibit significant changes. CONCLUSION: Moxibustion with acupuncture provided significant therapeutic benefits in patients with active CD beyond the placebo effect and is therefore an effective and safe treatment for active CD.

Protective effect of magnolol on lipopolysaccharide-induced acute lung injury in mice.

Magnolol, a tradition Chinese herb, displays an array of activities including antifungal, antibacterial, and antioxidant effects. To investigate the protective effect of magnolol on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in mice by intratracheal instillation of LPS (1 mg/kg). The mice received intratracheal instillation of magnolol (5 µg/kg) 30 min before LPS administration. Pulmonary histological changes were evaluated by hematoxylin-eosin stain and lung wet/dry weight ratios were observed. Concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and myeloperoxidase (MPO) activity were measured by enzyme-linked immunosorbent assay. Expression of cyclooxygenase (COX)-2 in lung tissues was determined by Western blot analysis. Magnolol pretreatment significantly attenuated the severity of lung injury and inhibited the production of TNF-α and IL-1ß in mice with ALI. After LPS administration, the lung wet/dry weight ratios, as an index of lung edema, and MPO activity were also markedly reduced by magnolol pretreatment. The expression of COX-2 was significantly suppressed by magnolol pretreatment. Magnolol potently protected against LPS-induced ALI and the protective effects of magnolol may attribute partly to the suppression of COX-2 expression.

[An experimental study on chemoprevention of esophageal adenocarcinoma by celecoxib, a selective cyclooxygenase-2 inhibitor].

OBJECTIVE: To study the feasibility of chemoprevention of esophageal adenocarcinoma by celecoxib, a selective cyclooxygenase-2(COX-2) inhibitor using a rat model. METHODS: Rats were divided into 3 groups: model group, celecoxib group, and control group. The rat surgical model was established by performing a gastrojejunostomy plus an esophagojejunostomy 5 mm distal to the gastrojejunal anastomosis. Twenty-eight weeks after surgery, all the animals were sacrificed and the pathological changes in the esophagus were examined macroscopically. COX-2 expression was analyzed by immunohistochemistry. Prostaglandin E2(PGE2) level was measured by enzyme-linked immunosorbent assay(ELISA). RESULTS: The incidence of Barrett's esophagus and esophageal adenocarcinoma in the model group was 84% and 57% respectively, significantly higher than those in the control group(P<0.01). The incidence of esophageal adenocarcinoma in the celecoxib-treated group was significantly lower than that in the model group(P<0.01), and no esophageal adenocarcinoma was detected in the control group. COX-2 expression was detected in 100% of reflux esophagitis, Barrett esophagus and esophageal adenocarcinoma, but not found in the normal tissue from the esophagus and the jejunum(P<0.01). The PGE2 level in the esophageal tissue in the model group was significantly higher than that in the control group(P<0.01). Rats in the celecoxib-treated group had significantly lower PGE2 level than that in the model group(P<0.01). The PGE2 levels were significantly higher in rats with cancer than those without cancer(P<0.01). CONCLUSION: Celecoxib successfully prevents the development of esophageal adenocarcinoma in a rat surgical model with mixed reflux of acid and duodenal juice and significantly decreases the risk of Barrett esophagus developing esophageal adenocarcinoma. COX-2 maybe an effective selective target of chemoprevention for esophageal adenocarcinoma.

[Study on inter-individual variability of cerebral response to acupuncture with fMRI].

OBJECTIVE: Effects of inter-individual variability on fMRI of acupuncture were observed and the possible influencing factors were further analyzed. METHODS: Twenty-six healthy volunteers were selected. And acupuncture was applied at Zusanli (ST 36) on the left side with even manipulation. The same experimental designation and data collecting reference were adopted to collect functional data. Then, the same data processing method was applied for analyzing individual data. Data which did not confirm with data analyzing qualification were rejected. The 26 individual data which met the requirement were taken randomly for 5 times according to the principle of random group division. Five groups named with A, B, C, D and E were thus generated with 11 samples in each. Images were processed with the AFNI software for every group, and the activated brain areas were revealed. RESULTS: Activated areas in the brain were observed in all the 5 groups, and the results vary a lot among different groups. Decreased signals of activated brain areas were observed in group C, while increased signals were seen in group D. Partial increasing and partial decreasing signals appeared in the other 3 groups. Compared with other groups, group D demonstrated totally different activated areas. The rate of difference among different groups is 46.7%-100.0%, and most of the differences were over half of the activated areas. CONCLUSION: Under the pre-requisites of strict control of experimental designation, acupuncture method, data collecting and processing, great differences have been found in the activated areas of the brain. It indicates that obvious individual differences existes in the activated areas of the brain with acupuncture. And the difference may greatly influence the researching result of fMRI as well as conclusions of those results.

[Experimental study on acute respiratory distress syndrome and analysis of relevant factors in rabbits subjected to thoracic blast trauma].

OBJECTIVE: To reproduce acute respiratory distress syndrome (ARDS) model in rabbit induced by chest blast injury and to analyze the pathogenesis and causes of early death in order to provide the basis for the early diagnosis of lung blast injury and its early warning system to facilitate an early treatment. METHODS: Sixty healthy New Zealand white rabbits were divided into six groups according to the different explosion distance with the random number table method. The survival rate and its resulting pathological changes were observed and patho physiological indexes and lung fluid content were determined at sequential time points post explosion. RESULTS: Shock wave pressure less than 1 210.5 mm Hg (1 mm Hg=0.133 kPa, group A, B) resulted in limited injury to the lung within grade 2 as assessed with the abbreviated injury scale (AIS). The rabbits in these groups recovered soon and survived without any complication. Shock pressure higher than 2 036.1 mm Hg (group D, E) caused severe injuries to the lung, including deep laceration , disruption of lung hilus and large hematoma in the lung, and the injury severity of lungs was assessed above grade 5 as assessed with AIS. All rabbits died within 1 hour post explosion. The groups described above failed to meet the demand of an ARDS model for the present study. Shock wave pressure at 1 917.3 mm Hg (group C) produced extensive contusion from grade 4 to grade 5 as assessed with AIS. The rabbits survived in poor general condition, and arterial partial pressure of oxygen (PaO(2)) lowered within 6 hours . Pathological examination showed extensive and constant multi focal bleeding involving more than four lobes. The alveolar wall was edematous, with partial rupture and alveolar fusion in lung tissues was observed in the group C. Alveoli were filled with inflammatory cells, and hyaline membrane was formed occasionally . Compared with control group, the wet to dry weight ratio (W/D) in lungs increased obviously (6.46±0.24 vs. 3.98±0.19, P<0.01) in group C within 6 hours postinjury. The contents of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in plasma and bronchoalveolar lavage fluid (BALF) were also increased distinctly compared with the control group [TNF-α (ng/L) in plasma: 328.89±6.26 vs. 62.12±2.98, TNF-α (ng/L) in BALF: 164.87±4.59 vs. 29.51±1.12; IL-6 (ng/L) in plasma: 128.51±4.13 vs. 19.32±1.53, IL-6 (ng/L) in BALF: 94.97±1.14 vs. 22.72±0.19, all P<0.05]. CONCLUSION: In an airtight environment, rabbit ARDS model can be reproduced successfully by blast injury with 1 917.3 mm Hg explosion pressure; TNF-α and IL-6 are involved in the pathogenesis and development of ARDS in blast injury. Pneumothorax as a result of lung rupture is the chief reason for early death and dysfunction of circulatory system is also an important reason in producing early death.

[Mechanism of As(2)O(3) on hdpr1 gene demethylation in Jurkat cell line].

This study was purposed to investigate the effect of As(2)O(3) on the demethylation of anti-oncogene-hdpr1 gene of acute lymphoblastic leukemia cell line Jurkat in vitro and its mechanism. The inhibitory effect of As(2)O(3) on the proliferation of Jurkat cells was assayed by CCK-8; the change of Jurkat cell cycle was detected by flow cytometry before and after using As(2)O(3); the effect of As(2)O(3) on the methylation model of hdpr1 gene was analyzed by methylation-specific PCR, and the effect of As(2)O(3) on the expression of hdpr1 mRNA was analyzed by semiquantitative RT-PCR. The results showed that the proliferation rate of Jurkat cells was decreased significantly after being treated with As(2)O(3), and in dose-and time-dependent manner; As(2)O(3) blocked Jurkat cell cycle in G(0)/G(1) phase in dose-dependent manner. As(2)O(3) could reverse hypermethylation of hdpr1 gene and induce its mRNA reexpression, and down-regulate the dnmt1, dnmt3a, dnmt3b mRNA expression level also in dose-dependent manner. It is concluded that the As(2)O(3) suppresses the proliferation of Jurkat cells and blocks cell cycle is G(0)/G(1), its possible mechanism may be down-regulating mRNA expression level of dnmt1, dnmt3a and dnmt3b, induce demethylation of hdpr1 gene from abnormal hypermethylation status and activates its reexpression.

[The significance of methylation status of secreted frizzled-related protein gene promoter in acute leukemia].

OBJECTIVE: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) gene and acute leukemia (AL). METHODS: We examined the promoter methylation status of SFRP1, 2, 4 and 5 in primary or relapsed AL patients, cell lines (HL60, NB4, Molt-4 and Jurkat) and peripheral blood mononuclear cells from healthy people with methylation-specific PCR (MSP). RESULTS: None of the normal mononuclear cells showed methylation of any SFRP genes. The frequencies of aberrant methylation among the samples were 33.9% (20/59) for SFRP1, 23.7% (14/59) for SFRP2, 6.8% (4/59) for SFRP4 and 10.2% (6/59) for SFRP5 in acute myelocytic leukemia (AML), and 39.3% (11/28) for SFRP1, 28.6% (8/28) for SFRP2, 25.0% (7/28) for SFRP4 and 32.1% (9/28) for SFRP5 in acute lymphoblastic leukemia (ALL). Hypermethylation of SFRP1, 2, 5 genes were present in the 4 AL cell lines. SFRP4 was methylated in NB4, Molt-4 and Jurkat cell lines. However, methylation and unmethylation of SFRP4 were both detected in HL60. CONCLUSIONS: Hypermethylation of SFRP genes is a common event in the evolution of AL. Methylation of SFRP genes might serve as potential independent biomarkers for early detection of AL.

[Inhibitory effect of VPA on multiple myeloma U266 cell proliferation and regulation of histone acetylation].

This study was aimed to investigate the effects of sodium valproate (VPA) on the proliferation and regulation of histone acetylation of multiple myeloma cell line U266. U266 cells were treated with VPA. Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). The expression level of HDAC1 mRNA was detected by RT-PCR, and the protein levels of HDAC1 and histone H3, H4 acetylation was detected by Western blot. The results showed that the VPA inhibited the proliferation of U266 cells in concentration-and time-dependent manners.After exposure to different concentrations of VPA for 48 hours, the proportion of G(0)/G(1) cells increased, while the proportion of S phase cells decreased. The cell cycle was arrested obviously in G(0)/G(1) phase (p < 0.05). The expression of HDAC1 mRNA was inhibited, and the protein level of HDAC1 was down-regulated, while the histone H3/H4 acetylation was up-regulated in U266 cells. It is concluded that the VPA can inhibit cell proliferation of U266 and induce G(0)/G(1) phase arrest. The increase of histone H3/H4 acetylation resulting from inhibiting HDAC1 by VPA might be considered as a possible mechanism.

[Investigation on the mechanisms of p15INK4B gene demethylation by valproate in Molt-4 cells].

OBJECTIVE: To study the antitumour effects of sodium valproate (VPA) on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms. METHODS: After Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 (DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR. RESULTS: VPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G(0)/G(1) phase was (66.87 ± 3.31)% and in S phase was (8.47 ± 2.56)%, while in control group, the cells in G(0)/G(1) phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration. CONCLUSION: VPA has a demethylation effect on p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G(0)/G(1) phase.

Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells.

Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.

[Methylation status of CpG islands in secreted frizzled-related protein gene promoter region of malignant hematopoietic cell lines].

To investigate the methylation status of CpG islands of the secreted frizzled-related protein (SFRP) gene promoter region in malignant hematopoietic cell lines, and to explore the possible relationship of CpG abnormal methylation status with pathogenic mechanism of hematologic malignancies. Methylation-specific PCR was used to detect the status of SFRP gene promoter region in nine malignant hematopoietic cell lines and peripheral blood mononuclear cells from healthy people. The results indicated that hypermethylation of 2 genes coding for SFRP1 and 2 were present in nine malignant hematopoietic cell lines, however, methylation and unmethylation of SFRP4 were both detected in CA46, HL60 and U937 cell lines, and SFRP5 in U266 as well. None of the normal mononuclear cells showed methylation of SFRP 1-5 genes. It is concluded that the hypermethylation of SFRP genes is related to the evolution of malignant hematopoiesis. Methylation of SFRP genes may serve as potential independent biomarkers for early detection of hematologic malignancies.

Concurrent expression of aryl hydrocarbon receptor and CYP1A1 but not CYP1A1 MspI polymorphism is correlated with gastric cancers raised in Dalian, China.

The frequency of cancer-associated m2m2- (C-) genotype of CYP1A1 and the factors contributing to the increased CYP1A1 expression in gastric cancers (GCs) are largely unknown. To address theses issues, PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to elucidate the MspI polymorphism in 60 GC cases and 57 normal donor samples. The frequencies of m1m1-, m1m2- and m2m2-genotype were 43.3, 45 and 11.7% among GC patients and 45.6, 49.1 and 5.3% among the normal donors respectively, demonstrating no significant difference of them between cancer and control groups (chi(2)=0.343, P=0.558). The correlation of Aryl hydrocarbon receptor (AhR) with the frequent CYP1A1 expression in stepwise gastrocarcinogenesis was determined by RT-PCR, immunohistochemical staining (IHC) and Western blotting, using GC samples as well as their pre-malignant and non-cancerous counterparts. RT-PCR revealed that the AhR detection rates were 100, 94.12 and 85.17% in GC, pre-malignant and non-cancerous mucosa (P>0.05) respectively but the level of AhR expression in GCs was much higher than that of non-cancerous tissues. IHC showed that the frequencies of AhR detection were 94.87% (37/39) in GCs, 94.12% (16/17) in pre-malignant lesions and 50% (3/6) in non-cancerous mucosa, revealing significant difference in frequencies of AhR detection and levels of AhR expression between GC or pre-malignant group and non-cancerous one (P<0.05). The frequency of AhR nucleus translocation was significantly high in GCs (94.87%; 37/39) than that in pre-malignant (70.59%; 12/17) and especially in non-cancerous group (16.67%; 1/6). Co-existence of AhR nuclear translocation and CYP1A1 expressions were found in 82.70% (43/52) of GCs (r(s)=0.437, P<0.01). Our results suggest (1) that CYP1A1 MspI polymorphism may not contribute to the high gastric cancer risk in Dalian region and (2) that enhanced AhR expression and especially its nuclear translocation may be a favorable factor for GC formation presumably via up-regulating CYP1A1 expression.