Functional analysis of flower development related gene GsLFY from Glycine soja.

LEAFY/FLORICAULA (LFY/FLO) is a family of plant-specific transcription factors, which plays an important role(s) in the regulation of floral organ formation and development. So far, LFY regulation on floral development in wild soybean has not been reported in the literature. In this study, the LFY gene, GsLFY, has been isolated from Glycine soja, and characterized with molecular and transgenic techniques. The cDNA for GsLFY gene is 1224 bp in length and contains an open reading frame encoding a polypeptide of 407 amino acids. Quantitative RT-PCR analysis shows that GsLFY is prominently expressed in various tissues, including roots, flowers and seeds. Among the four floral organs, GsLFY is expressed highly in sepals and stamens while weakly in the petals and carpels. Yeast two-hybrid experiments show that GsLFY possesses transactivation activity while transient expression analysis with Arabidopsis thaliana protoplasts shows that GsLFY protein is localized in the nucleus, supporting the notion that GsLFY is a transcription factor. The GsLFY transgenic tobacco plants flower about 29 days earlier than the wild-type plants, thereby providing a potential rationale for developing new soybean varieties with altered flowering time.

[Chemical constituents from Polygonum multiflorum].

Six compounds were isolated from the ethyl acetate extract of the tuberous root of Polygonum multiflorum by silical gel, ODS revsered-phase silica gel, Sephadex LH-20, and preparative HPLC. The structures were elucidated as column chromatography over： 2, 3, 5, 4'-tetrahydroxystilbene-2-O-(2″-O-p-hydroxybenzoyl)-ß-D-glucoside(1),2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucoside(2),2, 4, 6, 4'-tetrahydroxystilbene-2-O-ß-D-glucoside(3), emodin-8-O-ß-D-glucopyranoside(4), physcion-8-O-ß-D-glucopyranoside(5), and 8-O-methyl-emodin(6) on the basis of spectral analysis. Compound 1 is a new stilbene glycoside.

MicroRNA-30b functions as a tumour suppressor in human colorectal cancer by targeting KRAS, PIK3CD and BCL2.

Colorectal cancer (CRC) is the third most common cancer in the USA. MicroRNAs play important roles in the pathogenesis of CRC. In this study, we investigated the role of miR-30b in CRC and found that its expression was significantly lower in CRC tissues than that in normal tissues. We showed that a low expression level of miR-30b was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. Further experiments showed that over-expression of miR-30b suppressed CRC cell proliferation in vitro and tumour growth in vivo. Specifically, miR-30b promoted G1 arrest and induced apoptosis. Moreover, KRAS, PIK3CD and BCL2 were identified as direct and functional targets of miR-30b. MiR-30b directly targeted the 3'-untranslated regions of their mRNAs and repressed their expression. This study revealed functional and mechanistic links between miRNA-30b and oncogene KRAS, PIK3CD and BCL2 in the pathogenesis of CRC. MiR-30b not only plays important roles in the regulation of cell proliferation and tumour growth in CRC, but is also a potential prognostic marker or therapeutic target for CRC. Restoration of miR-30b expression may represent a promising therapeutic approach for targeting malignant CRC.

Metastasis-Associated in Colon Cancer-1 Associates With Poor Prognosis and Promotes Cell Invasion and Angiogenesis in Human Cervical Cancer.

OBJECTIVE: The aim of this study is to investigate the clinicopathologic significance and potential role of metastasis-associated in colon cancer-1 (MACC1) in the progression of cervical cancer. METHODS: MACC1 expression was examined in cervical cancer cell lines, 6 matched cervical cancer tissues, and adjacent noncancerous tissues using Western blotting and real-time reverse transcriptase polymerase chain reaction. MACC1 protein expression and localization were determined in 181 paraffin-embedded archived cervical cancer samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. The effects of MACC1 on cell migration, invasion, and angiogenesis were examined using migration assay, wound healing assay, 3-dimensional morphogenesis assay, and chicken chorioallantoic membrane assay. Western blotting was performed to examine the impact of MACC1 on the Akt and nuclear factor κB signaling pathways. RESULTS: Both protein and messenger RNA levels of MACC1 was up-regulated in cervical cancer cell lines and cervical cancer tissues, as compared with normal tissues. High MACC1 expression was detected in 96 (53%) of 181 of the cervical cancer tissues. In addition, high MACC1 expression correlated significantly with aggressiveness of cervical cancer, including International Federation of Gynecology and Obstetric stage (P = 0.001), pelvic lymph node metastasis (P = 0.004), recurrence (P = 0.037), and poor survival (P = 0.001). Moreover, enforced expression of MACC1 in cervical cancer cell lines significantly enhanced cell migration, invasion, and angiogenesis. Conversely, knockdown of MACC1 caused an inhibition of cell migration, invasion, and angiogenesis. Up-regulation of MACC1 increased, but knockdown of MACC1 decreased the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, enforced expression of MACC1 could enhance, but knockdown of MACC1 could reduce AKT and nuclear factor κB pathway activity. CONCLUSIONS: Our findings suggest that MACC1 protein, as a valuable marker of cervical cancer prognosis, plays an important role in the progression of human cervical cancer cells.

High expression level and nuclear localization of Sam68 are associated with progression and poor prognosis in colorectal cancer.

BACKGROUND: Src-associated in mitosis (Sam68; 68 kDa) has been implicated in the oncogenesis and progression of several human cancers. The aim of this study was to investigate the clinicopathologic significance of Sam68 expression and its subcellular localization in colorectal cancer (CRC). METHODS: Sam68 expression was examined in CRC cell lines, nine matched CRC tissues and adjacent noncancerous tissues using reverse transcription (RT)-PCR, quantitative RT-PCR and Western blotting. Sam68 protein expression and localization were determined in 224 paraffin-embedded archived CRC samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. RESULTS: Sam68 was upregulated in CRC cell lines and CRC, as compared with normal tissues; high Sam68 expression was detected in 120/224 (53.6%) of the CRC tissues. High Sam68 expression correlated significantly with poor differentiation (P = 0.033), advanced T stage (P < 0.001), N stage (P = 0.023) and distant metastasis (P = 0.033). Sam68 nuclear localization correlated significantly with poor differentiation (P = 0.002) and T stage (P =0.021). Patients with high Sam68 expression or Sam68 nuclear localization had poorer overall survival than patients with low Sam68 expression or Sam68 cytoplasmic localization. Patients with high Sam68 expression had a higher risk of recurrence than those with low Sam68 expression. CONCLUSIONS: Overexpression of Sam68 correlated highly with cancer progression and poor differentiation in CRC. High Sam68 expression and Sam68 nuclear localization were associated with poorer overall survival.

[Survey on pinworm infection and egg contamination among urban, suburban and rural pupils in Shangqiu City].

Seven hundred and ninety-eight preschool children and grade one pupils from three schools in the city of Shangqiu were sampled randomly in urban, suburban and rural areas. The transparent tape method was used to examine the infection of pinworm and the contamination of pinworm eggs on the environments. The average infection rate of pinworm was 9.9% (79/798). The prevalence of pinworm infection among the pupils of urban(4.6%) was statistically lower than those of suburban (11.2%) and rural (13.8%) (P < 0.01). The contamination rate of pinworm eggs from armor, fingers, bedclothes, briefs,and stationery in infected pupils are 23.8% (5/21), 18.0% (9/50), 15.8% (3/19), 12.9% (4/31) and 5.0% (2/40), respectively, which showed no statistical significance (P > 0.05).

Flavans from Iris tenuifolia and their effects on ß-amyloid aggregation and neural stem cells proliferation in vitro.

Two new flavans (1, 2) and a new flavanone (3), together with three known compounds (4-6), were isolated from the roots of Iris tenuifolia. Their structures were elucidated by means of spectroscopic methods including 1D and 2D NMR techniques and mass spectrometry. Compounds 1, 4, and 6 were further confirmed by single-crystal X-ray diffraction analysis. Biological evaluation showed that compounds 1 and 4 were positive in inhibiting ß-amyloid (Aß) aggregation and promoting neural stem cells (NSCs) proliferation, respectively.

[Stable isotope ratio characteristics and origin tracing of Portunus trituberculatus]. / ä¸åŒäº§åœ°ä¸‰ç–£æ¢­å­èŸ¹çš„ç¨³å®šåŒä½ç´ æ¯”å€¼ç‰¹å¾åŠåŽŸäº§åœ°æº¯æº.

A method for geographical discrimination of Portunus trituberculatus was explored to provide technical support for the protection of geographical indication products and for tracing the origin of seafood. P. trituberculatus were collected from three major production areas, including the Yellow Sea, the Bohai Sea, and the East China Sea. The variations of carbon and nitrogen stable isotope values of origins and the correlation of stable isotope ratios in different tissues were analyzed. The results showed that there were significant differences in carbon and nitrogen stable isotope ratio among different origins. Significant isotope fractionation effects were observed among different tissues. The discriminant model was developed and the origin discriminant analysis was performed by the stable isotope ratios of different tissues in P. trituberculatus. The correct rate of origin diffe-rentiationf using carbon and nitrogen stable isotopes in muscle and gills (>95%) was significantly higher than that of hepatopancreas and gonad, indicating that stable isotope ratios of muscle and gills could effectively differentiate P. trituberculatus in different sea areas. This study filled the gap of stable isotope tracing technology for P. trituberculatus.

The Protective Role of Mitochondrial Ferritin on Erastin-Induced Ferroptosis.

Ferroptosis, a newly identified form of regulated cell death, is characterized by overwhelming iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Preventing cellular iron overload by reducing iron uptake and increasing iron storage may contribute to inhibit ferroptosis. Mitochondrial ferritin (FtMt) is an iron-storage protein that is located in the mitochondria, which has a significant role in modulating cellular iron metabolism. Recent studies showed that FtMt played inhibitory effects on oxidative stress-dependent neuronal cell damage. However, the potential role of FtMt in the progress of ferroptosis in neuronal cells has not been studied. To explore this, we established ferroptosis models of cell and drosophila by erastin treatment. We found that overexpression of FtMt in neuroblastoma SH-SY5Y cells significantly inhibited erastin-induced ferroptosis, which very likely was achieved by regulation of iron homeostasis. Upon erastin treatment, significant increases of cellular labile iron pool (LIP) and cytosolic ROS were observed in wild-type SH-SY5Y cells, but not in the FtMt-overexpressed cells. Consistent with that, the alterations of iron-related proteins in FtMt-overexpressed cells were different from that of the control cells. We further investigated the role of FtMt in erastin-induced ferroptosis in transgenic drosophila. We found that the wild-type drosophilas fed an erastin-containing diet didn't survive more than 3 weeks. In contrast, the FtMt overexpressing drosophilas fed the same diet were survival very well. These results indicated that FtMt played a protective role in erastin-induced ferroptosis.

TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways.

BACKGROUND: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/ß-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce. METHODS: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR. RESULTS: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1. CONCLUSION: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

FOXC2 promotes colorectal cancer proliferation through inhibition of FOXO3a and activation of MAPK and AKT signaling pathways.

Abnormal expression of FOXC2 has been found in several human cancers. However, the role of FOXC2 in the progression of colorectal cancer (CRC) has not been well characterized. In analysis of 206 CRC specimens, we revealed that both high expression and nuclear localization of FOXC2 were correlated to aggressive characteristics and poor survival of patients with CRC. FOXC2 promoted cell proliferation through activation of MAPK and AKT pathways, subsequently down-regulating p27, up-regulating cyclin D1 and p-FOXO3a. Furthermore, FOXC2 nuclear localization was required for its promotion of cell proliferation. These findings suggest that FOXC2 plays an essential role in CRC progression and may serve as a valuable clinical prognostic marker of this disease.

Tiam1 transgenic mice display increased tumor invasive and metastatic potential of colorectal cancer after 1,2-dimethylhydrazine treatment.

BACKGROUND: T lymphoma invasion and metastasis 1 (Tiam1) is a potential modifier of tumor development and progression. Our previous study in vitro and in nude mice suggested a promotion role of Tiam1 on invasion and metastasis of colorectal cancer (CRC). In the present study, we generated Tiam1/C1199-CopGFP transgenic mice to investigate the tumorigenetic, invasive and metastatic alterations in the colon and rectum of wild-type and Tiam1 transgenic mice under 1,2-dimethylhydrazine (DMH) treatment. METHODS: Transgenic mice were produced by the method of pronuclear microinlectlon. Whole-body fluorescence imaging (Lighttools, Edmonton, Alberta, Canada), PCR, and immunohistochemical techniques (IHC) were applied sequentially to identify the transgenic mice. The carcinogen DMH (20 mg/kg) was used to induce colorectal tumors though intraperitoneal (i.p.) injections once a week for 24 weeks from the age of 4 weeks on Tiam1 transgenic or non-transgenic mice. RESULTS: We successfully generated Tiam1/C1199-CopGFP transgenic mice and induced primary tumors in the intestine of both wild type and Tiam1 transgenic mice by DMH treatment. In addition, Tiam1 transgenic mice developed larger and more aggressive neoplasm than wild-type mice. Moreover, immunohistochemical staining revealed that upregulation of Tiam1 was correlated with increased expression of ß-Catenin and Vimentin, and downregulation of E-Cadherin in these mice. CONCLUSIONS: Our study has provided in vivo evidence supporting that Tiam1 promotes invasion and metastasis of CRC, most probably through activation of Wnt/ß-catenin signaling pathway, in a Tiam1 transgenic mouse model.

microRNA-224 promotes cell proliferation and tumor growth in human colorectal cancer by repressing PHLPP1 and PHLPP2.

PURPOSE: To investigate the clinicopathologic significance, role, and mechanism of action of microRNA-224 (miR-224) in colorectal cancer. EXPERIMENTAL DESIGN: Real-time PCR was used to quantify miR-224 expression. The association of miR-224 with the clinicopathologic features and survival was evaluated in 110 colorectal cancer patients. The role of miR-224 in colorectal cancer was investigated using in vitro and in vivo assays. Luciferase reporter assays were conducted to confirm target gene associations. RESULTS: miR-224 was overexpressed in colorectal cancer. High-level expression of miR-224 was significantly associated with an aggressive phenotype and poor prognosis. Overexpression of miR-224 promoted colorectal cancer cell proliferation in vitro and tumor growth in vivo. Specifically, miR-224 accelerated the G1-S phase transition through activation of AKT/FOXO3a signaling, downregulation of p21Cip1 and p27Kip1, and upregulation of cyclin D1. Moreover, both PH domain leucine-rich-repeats protein phosphatase 1 (PHLPP1) and PHLPP2, antagonists of PI3K/AKT signaling, were confirmed as bona fide targets of miR-224. miR-224 directly targeted the 3'-untranslated regions of the PHLPP1 and PHLPP2 mRNAs and repressed their expression. CONCLUSION: This study reveals functional and mechanistic links between miRNA-224 and the tumor suppressors PHLPP1 and PHLPP2 in the pathogenesis of colorectal cancer. miR-224 not only plays important roles in the regulation of cell proliferation and tumor growth in colorectal cancer, but also has potential as a prognostic marker or therapeutic target for colorectal cancer.

HOXB7 as a prognostic factor and mediator of colorectal cancer progression.

PURPOSE: This study was to investigate the clinicopathologic significance and potential role of HOXB7 in the development and progression of colorectal cancer (CRC). EXPERIMENTAL DESIGN: The relationship between HOXB7 expression and clinical characteristics of CRC was analyzed in 224 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of HOXB7 on cell growth and proliferation, as well as on tumorigenesis, were examined both in vitro and in vivo, using MTT assay, colony formation assay, cell cycle analysis, soft agar assay, and tumorigenesis in nude mice. Western blotting and real-time reverse transcriptase-PCR were performed to examine the impact of HOXB7 on the PI3K/Akt and MAPK signaling pathways. RESULTS: HOXB7 protein level was significantly correlated with advanced Dukes stage (P < 0.001), T stage (P = 0.012), distant metastasis (P = 0.042), higher proliferation index (P = 0.007) and poor survival of patients (P = 0.005). Enforced expression of HOXB7 in CRC cell lines significantly enhanced cell growth, proliferation and tumorigenesis. Conversely, knockdown of HOXB7 caused an inhibition of cell growth, proliferation, and tumorigenesis. We also showed that HOXB7 accelerated G(0)-G(1) to S-phase transition concomitantly with upregulation of cyclin D1 and downregulation of p27Kip1. On the contrary, knockdown of HOXB7 caused G(1)-S-phase arrest, downregulation of cyclin D1 and upregulation of p27Kip1. Enforced expression of HOXB7 could enhance PI3K/AKT and MAPK pathway activity. CONCLUSION: Our findings suggest that HOXB7 protein, as a valuable marker of CRC prognosis, plays an important role in the development and progression of human CRC.

Determination of trace trichlorfon by high performance liquid chromatography with UV detection based on its catalytic effect on sodium perborate oxidizing benzidine.

Trichlorfon has the capacity to catalyze the oxidation of benzidine (4,4'-diamino-biphenyl) to 4-amino-4'-nitro biphenyl in the presence of sodium perborate. The product of the catalyzed reaction was validated by LC-MS method. Reversed-phase high performance liquid chromatography with 365 nm UV detection was used for separation and quantification of 4-amino-4'-nitro biphenyl. It can be proven there is a linear relationship between the peak areas of 4-amino-4'-nitro biphenyl and trichlorfon in the concentration range of 0.02-0.5 mg L(-1) (r=0.9988). Limit of detection was 2.0 microg L(-1). A method for the indirect determination of trichlorfon using HPLC was developed based on catalytic effect of trichlorfon. Method validation was performed on samples spiked at three levels (0.5, 1.0, 1.5 mg kg(-1)), the recoveries ranged from 67.5 to 82.1%, with relative standard deviations between 4.5 and 7.3%. 0.01 mol L(-1) sodium dodecyl sulphate (SDS) solution was used to extract trichlorfon from samples and solid-phase extraction was used to isolate and concentrate trichlorfon in SDS solution. The recoveries of trichlorfon obtained with percolating the extraction through a SPE system were essentially in agreement with those obtained by liquid-liquid extraction. This new isolation technique decreases the use of toxic solvents and satisfies the requirements of Green Analytical Chemistry.