[miR-758-3p inhibits the proliferation and invasion of non-small cell lung cancer cells by targeting NUSAP1].

Objective: To investigate the biological function of miR-758-3p and the underlying mechanism in non-small cell lung cancer (NSCLC). Methods: miR-758-3p mimics was transfected to A549 NSCLC cells, miRNA control was used as a negative control, cells transfected with nucleolar and spindle associated protein 1 (NUSAP1)-overexpression vector indicated as NUSAP1 group, cells co-transfected with miR-758-3p mimics and NUSAP1-overexpression vector indicated as miR-758-3p mimics+ NUSAP1 group. The effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells were detected by cell counting kit-8 (CCK-8) and Transwell assays, respectively. Bioinformatics and dual luciferase reporter assay were used to predict and verify the target gene of miR-758-3p. Results: The expressions of miR-758-3p and NUSAP1 in A549 cells were significantly up-regulated at 24 hours after transfection with miR-758-3p mimics (P<0.05). Compared with the miRNA control group (1.15±0.06), the OD value of miR-758-3p mimic group (0.78±0.06) was significantly decreased at 72 hours after transfection (P<0.05). The number of migrated cells of miR-758-3p mimic group (119.04±11.49) was significantly lower than that of the control group (271.38±19.05) (P<0.05). The number of invaded cells of miR-758-3p mimic group (71.33±5.36) was significantly lower than the control group (164.30±8.11) (P<0.05). The result of dual-luciferase reporter assay showed that NUSAP1 was a direct target of miR-758-3p. Moreover, up-regulation of NUSAP1 abolished the inhibitory effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells. Conclusion: miR-758-3p inhibits the proliferation, migration and invasion of NSCLC cells by targeting NUSAP1.

MiR-210 inhibits apoptosis of cranial nerves in preeclampsia rats through suppressing the TGF-ß signaling pathway.

OBJECTIVE: To explore the protective effect of micro ribonucleic acid (miR)-210 on cranial nerves in rats with preeclampsia (PE) by regulating the transforming growth factor-ß (TGF-ß) signaling pathway. MATERIALS AND METHODS: A total of 36 pregnant Sprague-Dawley rats were randomly divided into normal group (n=12), model group (n=12), and miR-210 mimics group (n=12). The rats were fed normally in the normal group. In the latter two groups, the PE model was established, followed by injection of normal saline or miR-210 mimics via the caudal vein, respectively. The above intervention lasted until 20 d of gestational age in pregnant rats. Then, the systolic blood pressure of the caudal vein was measured. The relative levels of Caspase3, phosphorylated TGF-ß (p-TGF-ß), and miR-210 were detected via immunohistochemistry, Western blotting, and quantitative Polymerase Chain Reaction (qPCR). Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: The systolic blood pressure of the caudal vein significantly increased in the other two groups compared with that in the normal group (p<0.05), while it significantly decreased in the miR-210 mimics group compared with that in the model group (p<0.05). The results of immunohistochemistry showed that the positive expression of Caspase3 significantly rose in the other two groups compared with that in the normal group (p<0.05), while it remarkably declined in miR-210 mimics group compared with that in the model group (p<0.05). The results of Western blotting revealed that the protein expression of p-TGF-ß was evidently higher in the other two groups than that in the normal group (p<0.05), while it was evidently lower in the miR-210 mimics group than that in the model group (p<0.05). Moreover, it was found via qPCR that the other two groups had remarkably lower relative expression of miR-210 than normal group (p<0.05), while miR-210 mimics group had remarkably higher relative expression of miR-210 than the model group (p<0.05). According to the results of TUNEL assay, the apoptosis rate markedly increased in the other two groups compared with that in the normal group (p<0.05), while it markedly decreased in the miR-210 mimics group compared with that in the model group (p<0.05). CONCLUSIONS: MiR-210 inhibits apoptosis via suppressing the TGF-ß signaling pathway, thereby exerting a protective effect on cranial nerves in PE rats.

[Study on the damage of the tight junctions of nasal mucosal epithelial cells by artemisia annua pollen].

Objective: To investigate the damage and mechanism of artemisia annua pollen on tight junction of human nasal mucosa epithelial cells (HNEpC). Methods: HNEpC were cultured in vitro. Different concentrations of artemisia annua pollen (0, 20, 40, 80, 100, 160, 200 µg/ml) were used to intervene the cells for 24 h, and the cell proliferation activity was detected by the CCK-8 method. The expression and phosphorylation of p38MAPK signaling pathway were detected by Western Blot before and after the intervention of SB203580, a p38MAPK inhibitor in HNEpC. Immunofluorescence chemical staining, Western Blot and quantitative real-time PCR (qPCR) were used to observe the expression and distribution of tight junctions Occludin and Claudin-1. SPSS 21.1 software was used for statistical analysis. Results: CCK-8 results showed that, compared with the control group, the proliferation activity of HNEpC increased after 6 h intervention with different concentrations of artemisia annua pollen (all P<0.05). After 12 h of intervention, the proliferation activity of HNEpC in the 20, 40, 80, 100 and 160 µg/ml groups was not significantly changed (all P>0.05), while that in the 200 µg/ml group was decreased (P<0.05). After the intervention for 24 h, the proliferation activity of cells in the 20 and 40 µg/ml groups was not significantly changed (all P>0.05), while that in the 80, 100, 160 and 200 µg/ml groups was decreased (all P<0.05). Immunofluorescence staining showed that the Occludin and Claudin-1 proteins in the normal control group were localized on the cell membrane and expressed more and formed a ring structure around the cell membrane. However, under the intervention of high concentration artemisia annua pollen, its expression level decreased, appeared broken, fuzzy, and nonuniform distribution. Western Blot and qPCR results showed that after 24 h of intervention, the expression levels of HNEpC Claudin-1 protein and its mRNA in the pollen groups (40, 80, 100, 160, 200 µg/ml) of artemisia annua decreased compared with those of those of the control group (mRNA expression levels were 0.567±0.214, 0.443±0.109, 0.462±0.160, 0.497±0.134, 0.388±0.076 compared with 1.001±0.067, respectively, all P<0.05). However, the mRNA of Occludin protein and its mRNA only decreased in the 200 µg/ml treatment group (mRNA expression level was 0.631±0.109 compared with 1.016±0.026, P<0.05), while all the other treatment groups increased (mRNA expression levels were 1.258±0.134, 1.827±0.103, 2.429±0.077, 1.707±0.085, 1.477±0.066 compared with 1.016±0.026, respectively, all P<0.05). Western Blot showed that p-p38MAPK expression increased after intervention with 100, 160, 200 µg/ml artemisia annua pollen for 24 h. SB203580 could inhibit the decreasing expression of Occludin caused by artemisinin pollen (mRNA expression was 1.255±0.179 compared with 0.631±0.109, P<0.05), but had no effect on Claudin-1 protein expression. Conclusion: Pollen from artemisia annua may activate p38MAPK signaling pathway and destroy the close connection of HNEpC.

[NUSAP1 promotes lung cancer progression by activating AKT/mTOR signaling pathway].

Objective: To investigate the inhibitory effects of nucleolar and spindle associated protein 1 (NUSAP1) on lung cancer and the related mechanisms. Methods: A549 cells were transfected with NUSAP1 siRNA, the cell proliferation, migration and invasion, and apoptosis were detected by CCK8, Transwell and flow cytometry, respectively. Western blot was used to detect the expressions of apoptosis and AKT/mTOR signal pathway related proteins. Results: Compared with the negative control group, the proliferation [(0.610±0.058) vs (1.724±0.067), P<0.05], migration [(178.267±14.780) vs (272.464±36.232), P<0.05] and invasion [(73.527±6.617) vs (120.585±13.235), P<0.05] of NUSAP1 deleted A549 cells were significantly inhibited, while the apoptosis [(3.572±0.214)% vs (11.358±1.047)%, P<0.05] was significantly increased. The expressions of apoptosis related protein Bax and active-caspase 3 were increased (P<0.05), while the expressions of anti-apoptosis protein Bcl-2 and proliferation related protein P70, the phosphorylation levels of AKT and mTOR were reduced in NUSAP1 knockdown cells (P<0.05). Conclusion: NUSAP1 knockdown can inhibit the proliferation, migration and invasion, and promote the apoptosis of tumor cells through suppressing AKT/mTOR signaling pathway in lung cancer cells.

[Hybrid coronary revascularization for the treatment of coronary artery disease].

Hybrid coronary revascularization (HCR) combines the respective strengths of coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) for selected patients with multivessel coronary artery disease. HCR includes the minimally invasive direct coronary artery bypass of a left internal thoracic artery graft to the left anterior descending artery (LAD) with stenting of non-LAD lesions with PCI, in onestop or staged hybrid procedures.HCR can prove superiority to conventional CABG in clinical complications. Many available data suggests that HCR is feasible and safe in skilled hands. Further large-scale randomized controlled trials are in great need. HCR may become another more commonly used interventional strategy for certain patients with multivessel coronary artery disease.

Continued Elevation of Plasma IL-4 and IL-17 Predicts the Progression from VMC to DCM.

OBJECTIVE: To investigate plasma cytokines (interferon gamma, interleukin-4, and interleukin-17) in patients with viral myocarditis (VMC) and evaluate their predictive value in the progression from VMC to dilated cardiomyopathy (DCM). METHODS: A prospective, multicenter, observational study included 536 patients with newly diagnosed VMC admitted in cardiology departments of 24 tertiary super specialised university-affiliated hospitals in the China registry from January 2012 to June 2016. Demographics and clinical characteristics at baseline and after three months were collected, including laboratory blood tests, ECG, echocardiography, and drug treatment in each participating site. The plasma anti-viral antibodies (Abs), anti-heart autoimmune Abs, and cytokines were detected by ELISA. RESULTS: Of the 536 patients, 534 were included for analysis after two patients died in less than a month. The plasma levels of IFN-γ, IL-4, and IL-17 were continually higher in patients with incident DCM than in those without incident DCM at baseline, from the 1st month and the 3rd month; all had a P value of <0.0001. There was a positive correlation between IL-4 and LVEDd (r = 0.30, P < 0.0001) and between IL-17 and LVEDd (r = 0.11, P = 0.02). When all these covariates have entered the model simultaneously, elevated IL-4 and IL-17 were still significantly associated with DCM incidence. The RR (95% CI) of DCM incidence were 1.04 (1.02-1.06) for IL-4 and 5.24 (2.81-9.79) for IL-17. CONCLUSION: The continued elevation of plasma IL-4 and IL-17 in VMC patients were associated with a high incidence of DCM at three months, and these two cytokines were independent predictors for the progression from VMC to DCM.

[Expression of NUSAP1 and its relationship with prognosis in non-small cell lung cancer].

Objective: To investigate the expression of nucleolar and spindle-associated protein 1 (NUSAP1) in non-small cell lung cancer (NSCLC) and analyze its relationship with the prognosis of NSCLC patients. Methods: Real-time fluorescent quantitative PCR and immunohistochemical staining were performed to determine the expression of NUSAP1 in NSCLC tissues and adjacent tissues collected from hospital. The relationship between NUSAP1 expression and prognosis of NSCLC patients was analyzed by online database. Results: The expression level of NUSAP1 mRNA in tumor tissues was significantly higher than that of adjacent tissues (P<0.05). The high expression rate of NUSAP1 protein in NSCLC tissues was 58.0% (29/50), significantly higher than 22.0% (11/50) of adjacent tissues (P<0.05). The high expression of NUSAP1 protein in NSCLC tissues was closely correlated with tumor size, lymph node metastasis and TNM stage (P<0.05), but was not related to age and gender. The data showed that the expression level of NUSAP1 mRNA was inversely associated with the overall survival (OS) of NSCLC patients (P<0.001). The expression of NUSAP1 mRNA was significantly correlated with the pathological grade, clinical stage, gender, chemotherapy, smoking history, and histological type of NSCLC patients (P<0.05). Conclusions: The expression of NUSAP1 is up-regulated in NSCLC, which is correlated with the growth and development of NSCLC and prognosis of the patients. These results indicate that NUSAP1 can be used as a potential prognostic marker for NSCLC.

Characterization, culture medium optimization and antioxidant activity of an endophytic vitexin-producing fungus Dichotomopilus funicola Y3 from pigeon pea [Cajanus cajan (L.) Millsp.].

AIMS: The aim of this study was to characterize a fungal endophyte Y3 from pigeon pea (Cajanus cajan [L.] Millsp), as a novel producer of vitexin, and its culture medium optimization and antioxidant activity. METHODS AND RESULTS: The endophyte from the leaves of pigeon pea was identified as Dichotomopilus funicola by the morphological and molecular characteristics. The most important medium variables affecting vitexin production in liquid culture of D. funicola Y3 were screened by Plackett-Burman design, and three culture medium constituents (i.e. l-phenylalanine, salicylic acid and CuSO4 ·5H2 O) were identified to play significant roles in vitexin production. The most significant factors were further optimized using by central composite design with response surface methodology. The DPPH radical-scavenging assay indicated that fungal vitexin exhibited notable antioxidant activity with an EC50 value of 164 µg l-1 . CONCLUSIONS: First, a novel endophyte vitexin-producing Dichotomopilus funicola Y3 was isolated from pigeon pea (Cajanus cajan[L.] Millsp.). The maximum vitexin yield was obtained as 78·86 mg l-1 under the optimum culture medium constituents: 0·06 g l-1  l-phenylalanine, 0·21 g l-1 salicylic acid, and 0·19 g l-1 CuSO4 ·5H2 O in medium, which is 4·59-fold higher than that in the unoptimized medium. Also, fungal vitexin clearly demonstrated its antioxidant potential. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings provide an alternative source for large-scale production of vitexin by endophytic fungal fermentation and have a promising prospect in food and pharmaceutical industry.

Unrecognised IgG4 association in progressively transformed germinal centers of lymph nodes with subsequent full-blown IgG4-related chronic fibrosing pancreatitis: A case report.

Progressively transformed germinal centers (PTGC) is a benign process characterised by a morphological variant of reactive follicular hyperplasia in lymph nodes. It was recently shown that some cases of PTGC are associated with IgG4-related disease (IgG4-RD) or increased IgG4 plasma cells. Five years ago, a 57-year-old woman presented with enlargement of multiple lymph nodes in the left parotid, submandibular, and neck areas, pathologically diagnosed as PTGC after excisional biopsy. Since then, she has experienced numbness in her extremities, especially the left shoulder and arm, pruritus on the left side of the face and intermittent facial palsy, for which she has been receiving regular symptomatic treatment. Recently the patient developed diabetes mellitus (approximately seven months ago). In routine follow-up scans, a mass was detected in left kidney and magnetic resonance imaging of the abdomen prior to surgery revealed a slightly enhanced bulky mass replacing the pancreatic tail and uncinate process. The mass in left kidney was diagnosed as clear cell renal cell carcinoma, and the pathological features of the pancreatic lesion were those of IgG4-related chronic fibrosing pancreatitis. Retrograde examination of the neck lymph node diagnosed as PTGC showed increased deposition of IgG4-positive plasma cells.

Effect of dietary fiber levels on performance, gizzard development, intestinal morphology, and nutrient utilization in meat ducks from 1 to 21 days of age.

The objective of this experiment was to determine the effects of dietary fiber levels on growth performance, gizzard development, intestinal morphology, and nutrient utilization in Cherry Valley meat ducks. In total, 720 1-day-old ducklings were fed with starter diets (120 ducklings, 8 pens of 15 ducklings, on each diet) containing 6 levels of crude fiber (CF, 1.46, 3.09, 4.15, 6.18, 7.52, and 9.03%, based on analysis) for 21 d. Then, on d 22, ducks fed all of the starter diets were transferred to a grower diet (containing 4% CF) to examine the residual effect of starter dietary fiber levels until 35 d. Body weight (BW), body weight gain (BWG), feed intake (FI), feed to gain ratio (F:G) were recorded/calculated weekly. Ducks were sampled for gizzard development and intestinal morphology determination on d 7, 14, and 21. Nutrient utilization was assessed using 25- to 27-day-old ducks. The results showed that BW (d 21), BWG (d 15 to 21, d 1 to 21), and F:G (d 15 to 21, d 1 to 21, and d 1 to 35) were increased quadratically (P < 0.01), and FI (all periods except for d 22 to 35) was increased linearly (P < 0.01), when starter dietary CF levels increased from 1.46 to 9.03%. Ducks under 3.09% and 4.15% CF starter diets had decreased 1 to 35 d FI and F:G when compared to ducks under other starter diets. When compared to ducks fed 1.46% and 3.09% CF starter diets, ducks fed starter diet containing 7.52% CF had increased gizzard development, jejunal morphology, energy retention, excreta nutrients availability, and standardized ileal digestibility (SID) of Arg, Ile, Leu, Thr, Val, Asp, Ala, Glu, Gly, Pro, Ser, and total amino acids. In conclusion, meat ducks from 1 to 21 d of age could adapt to a wide range (3.09% to 7.52%) of dietary fiber levels.

First-Line Use of Core Needle Biopsy for High-Yield Preliminary Diagnosis of Thyroid Nodules.

BACKGROUND AND PURPOSE: Although core needle biopsy was introduced as a diagnostic alternative to fine-needle aspiration, the utility and safety of core needle biopsy for thyroid nodules in a large population has yet to be studied comprehensively. We evaluate core needle biopsy yields on a large-scale basis to investigate its potential in the preliminary diagnosis of thyroid nodules. MATERIALS AND METHODS: Between March 2005 and December 2013, 2448 initially detected thyroid nodules from 2120 consecutive patients who underwent core needle biopsy were retrospectively evaluated. Of these, 72 thyroid nodules from 63 patients were excluded due to prior fine-needle aspiration attempts. The inconclusive and conclusive result rates, diagnostic accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and unnecessary surgery rate of core needle biopsy were evaluated. RESULTS: With core needle biopsy as the first-line method, the inconclusive result rate was 11.9% (283/2376) and the conclusive result rate was 88.1% (2093/2376). The diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of core needle biopsy for the diagnosis of malignancy were 96.7% (1160/1200), 89.7% (347/387), 100% (813/813), 100% (347/347), and 95.3% (813/853), respectively. There were no major complications and 12 minor complications. CONCLUSIONS: We have demonstrated that first-line use of core needle biopsy may well improve diagnostic accuracy in thyroid nodules, reducing inconclusive or false-negative results and unnecessary operations. Such benefits underscore the promising role of core needle biopsy in managing thyroid nodules and optimizing related surgical decision-making.

Attentional bias modulation by reappraisal in patients with generalized anxiety disorder: an event-related potential study

Affective states influence subsequent attention allocation. We evaluated emotional negativity bias modulation by reappraisal in patients with generalized anxiety disorder (GAD) relative to normal controls. Event-related potential (ERP) recordings were obtained, and changes in P200 and P300 amplitudes in response to negative or neutral words were noted after decreasing negative emotion or establishing a neutral condition. We found that in GAD patients only, the mean P200 amplitude after negative word presentation was much higher than after the presentation of neutral words. In normal controls, after downregulation of negative emotion, the mean P300 amplitude in response to negative words was much lower than after neutral words, and this was significant in both the left and right regions. In GAD patients, the negative bias remained prominent and was not affected by reappraisal at the early stage. Reappraisal was observed to have a lateralized effect at the late stage.

Attentional bias modulation by reappraisal in patients with generalized anxiety disorder: an event-related potential study.

Affective states influence subsequent attention allocation. We evaluated emotional negativity bias modulation by reappraisal in patients with generalized anxiety disorder (GAD) relative to normal controls. Event-related potential (ERP) recordings were obtained, and changes in P200 and P300 amplitudes in response to negative or neutral words were noted after decreasing negative emotion or establishing a neutral condition. We found that in GAD patients only, the mean P200 amplitude after negative word presentation was much higher than after the presentation of neutral words. In normal controls, after downregulation of negative emotion, the mean P300 amplitude in response to negative words was much lower than after neutral words, and this was significant in both the left and right regions. In GAD patients, the negative bias remained prominent and was not affected by reappraisal at the early stage. Reappraisal was observed to have a lateralized effect at the late stage.

Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella.

The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.

Two polymorphisms of RCAN1 gene associated with Alzheimer's disease in the Chinese Han population.

OBJECTIVE: Regulator of calcineurin 1 (RCAN1) gene is a regulator on the activity of calcineurin and was reported to be overexpressed in Alzheimer's disease. The aim of this study was to evaluate several polymorphisms of RCAN1, located in the probable promoter region of RCAN1-4 and around the exonic splicing enhancer motifs of RCAN1, in a cohort of Chinese late-onset Alzheimer's disease. METHODS: A pilot case-control study was conducted in 142 Alzheimer's disease patients and 99 nondemented controls from Chinese Han population. Fragments of the RCAN1 including 5 polymorphisms (rs71324311, rs3831376, rs10550296, rs8135540, rs78899361) were amplified and sequenced. RESULTS: In our sample, 2 polymorphisms (rs71324311 and rs10550296) were associated with Alzheimer's disease. Of these 2 polymorphisms, the heterozygous deletion genotype of rs71324311 was more prevalent in non-demented controls than in those with Alzheimer's disease (4% vs. 0%), indicating a slight protective role (Fisher's exact test, p = 0.03; crude odds ratio = 0.96, 95% confidence interval = 0.92-0.99). There was only a trend towards a significant difference in the distributions of genotypes of rs10550296 between 2 groups (chi2 = 1.93; p = 0.17; crude odds ratio = 1.44, 95% confidence interval = 0.85-2.41). However, logistic regression analysis showed that the age-, gender- and apolipoprotein E epsilon4-adjusted odds ratio of Alzheimer's disease with rs10550296 heterozygous deletion genotype was 2.11 (chi2 = 4.42; p = 0.04; 95% confidence interval = 1.05-4.20). CONCLUSIONS: Regarding Alzheimer's disease susceptibility in Chinese Han population, our data suggested a protective role for the rs71324311 heterozygous deletion genotype and a risk role from the rs10550296 heterozygous deletion genotype.

Gamma-tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways.

Tocotrienol-rich fraction (TRF) has demonstrated antiproliferative effect on prostate cancer (PCa) cells. To elucidate this anticancer property in PCa cells, this study aimed, first, to identify the most potent isomer for eliminating PCa cells; and second, to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the presence of sub-G(1) cell population. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R and Id family proteins (Id1 and Id3). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK-signalling pathway and inhibition of JNK activity by a specific inhibitor (SP600125) was able to partially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, a synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Our results suggested that the antiproliferative effect of gamma-tocotrienol act through multiple-signalling pathways, and demonstrated for the first time the anti-invasion and chemosensitisation effect of gamma-tocotrienol against PCa cells.

Activation and inactivation of horseradish peroxidase by cobalt ions.

The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.

Id-1 promotes chromosomal instability through modification of APC/C activity during mitosis in response to microtubule disruption.

Id-1 (Inhibitor of DNA binding/differential-1) plays a positive role in tumorigenesis through regulation of multiple signaling pathways. Recently, it is suggested that upregulation of Id-1 in cancer cells promotes chromosomal instability. However, the underlying molecular mechanism is not known. In this study, we report a novel function of Id-1 in regulation of mitosis through physical interaction with Cdc20 (cell division cycle protein 20) and Cdh1 (Cdc20 homolog 1). During early mitosis, Id-1 interacts with Cdc20 and RASSF1A (Ras association domain family 1A), leading to enhanced APC(Cdc20) activity, which in turn promotes cyclin B1/securin degradation and premature mitosis. During late mitosis, Id-1 binds to Cdh1 and disrupts the interaction between Cdh1 and APC, resulting in suppression of APC(Cdh1) activity. On the other hand, overexpression of Cdh1 leads to Id-1 protein degradation, suggesting that Id-1 may also act as a substrate of APC(Cdh1). The negative effect of Id-1 on APC(Cdh1) results in suppression of APC(Cdh1)-induced Aurora A and Cdc20 degradation, leading to failure in cytokinesis. As a result, overexpression of Id-1 in human prostate epithelial cells leads to polyploidy in response to microtubule disruption, and this effect is abolished when Id-1 expression is suppressed using antisense technology. These results demonstrate a novel function of Id-1 in promoting chromosomal instability through modification of APC/C activity during mitosis and provide a novel molecular mechanism accounted for the function of Id-1 as an oncogene.

Down-regulation of prostate specific antigen in LNCaP cells by flavonoids from the pollen of Brassica napus L.

The pollen of Brassica napus L. has been used in China to treat benign prostatic hyperplasia (BPH) for over decades. In this study, the pollen of Brassica napus L. was extracted successively with chloroform, ethyl acetate and ethanol. The ethyl acetate extract showed strong activity in decreasing the secretion of prostate specific antigen (PSA) in LNCaP cells as compared to two other extracts, measured by ELISA with finasteride as positive control in the assay. Five flavonoids were subsequently isolated from the active extract using bioassay-guided fractionation. They were Naringenin (1); Luteolin (2); Kaempferol (3); Kaempferol 3-(3-E-p-coumaroyl-alpha-L-rhamnopyranoside) (4); and Kaempferol 3-(2,3-di-E-p-coumaroyl-alpha-L-rhamnopyranoside) (5). All these compounds inhibited PSA secretion significantly, with IC50 values in the range of 5-50 microM. Compounds 2, 4 and 5 showed moderate cytotoxicity to LNCaP cells within the active concentration range, while compounds 1 and 3 showed no cytotoxicity. Further studies on the mechanism action of these compounds were performed by evaluating their activation of estrogen receptor (ER) and antagonistic activities on androgen receptor (AR) in cell-based reporter gene assays. All compounds described here were first isolated from the pollen of Brassica napus L.