Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. V_max, K_cat and K_m values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.
Cloning, Molecular ; Escherichia coli/metabolism* ; Phenylalanine/metabolism* ; Phenylalanine Ammonia-Lyase/chemistry* ; Recombinant Proteins ; Schisandra/genetics*

Professor
Acupuncture ; Acupuncture Therapy ; Angina, Stable ; Combined Modality Therapy ; Humans ; Moxibustion

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer.However,the exact relationship between them is still unknown.The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer.RNA interference technology was employed to silence filamin A in MDA-MB-231 cells.Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels,respectively.Double immunofluorescence was applied to show their colocalization morphologically.Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells.The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ.In addition,double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells.Silencing filamin A led to the enhanced fluorescence of 14-3-3σ.Furthermore,cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro.In conclusion,silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Objective To explore the effects of Cigu Xiaozhi Pills on lipotoxicity and oxidative stress in rats with non-alcoholic steatohepatitis (NASH); To discuss relevant mechanism of action. Methods SD rats were divided into six groups randomly:normal control group,model group,positive medicine group,Cigu Xiaozhi Pills high-,medium-, and low-dose groups. NASH model was established by feeding rats with high fat diet for 12 weeks. At the same time, the model rats were given medicine intervention. At the end of 12 weeks, all the experimental animals were killed and the liver and serum were taken. Serum samples were taken for detection of ALT, AST, TG, TC, T-SOD, LDL-C, MDA, GSH-Px and FFA. Liver tissues were taken for detection of T-SOD, MDA, GSH-Px and FFA. The liver histopathological changes were observed under microscope with HE staining. The ultrastructure of liver cells was observed by transmission electron microscope. The fatty degeneration of liver cells was observed by oil red O staining. Results Liver histopathological examination showed that the liver tissue of model group showed moderate to severe steatosis and inflammatory cell infiltration. Compared with normal control group, rat liver wet weight, liver index, ALT, AST, TG, TC, LDL-C, MDA and FFA in serum, and FFA and MDA in liver homogenate in model group significantly increased (P<0.05, P<0.01), while T-SOD and GSH-Px activity in serum and liver homogenate significantly decreased (P<0.05, P<0.01). Compared with model group, Cigu Xiaozhi Pills high-dose group could significantly decrease the elevation of serum ALT, AST, TG, TC, LDL-C, MDA and FFA (P<0.05, P<0.01), but increase T-SOD and GSH-Px activity in serum and liver tissue (P<0.01). The pathological section showed that:compared with model group, the hepatic lobule vacuolar degeneration and fatty degeneration were significantly reduced in Cigu Xiaozhi Pills high-, medium- and low-dose groups, and the inflammatory cell infiltration was improved. Conclusion Cigu Xiaozhi Pills can obviously improve liver function and blood lipid of NASH rat model induced by high-fat diet, enhance antioxidant capacity, reduce lipid peroxidation and achieve the purpose of prevention and treatment of NASH.

Purpose To detect Wnt5a and Wnt11 expression in lung cancer,to explore the relationship between their expression and the types of lung cancer,and to assess the relationships between their expression and clinicopathologic factors (such as gender,age,degree of cell differentiation,lymph node metastasis).Methods The 120 cases of lung cancer were selected as the experimental group.In addition,20 cases of normal lung tissue were selected as the control group.Immunohistochemistry EnVision method was used to detect the expression of Wnt5a and Wnt11.The results were analyzed by the statistical software.Results The positive expression rate of Wnt5a and Wnt1 1 was 36％ and 38％ in 84 cases of non-small cell lung carcinoma.While the expression rate of Wnt5a and Wnt11 were low (8.3％ and 0,respectively)in 36 cases of small cell lung carcinoma.Both expression in non-small cell lung carcinoma was significantly higher than that of small cell carcinoma.The expression rate of Wnt11 in adenocarcinoma was higher (56％,while Wnt5a was 9％),and the expression rate of Wnt5a in squamous cell carcinoma was higher (50％,while Wnt11 was 25％).However,there was no significant difference between the two groups in the sex,age,differentiation and lymph node metastasis.Conclusion Both Wnt5a and Wnt11 expression was associated with lung cancer types.The positive expression rate of Wnt5a and Wnt11 in non-small cell lung carcinoma is significantly higher than that of small cell lung carcinoma.The expression level of Wnt5a is higher in squamous cell carcinoma,while Wnt11 is higher in adenocarcinoma.Both of their expression show no significant correlation with the lung cancer clinicopathological indicators (including the gender,age,degree of differentiation and lymph node metastasis in patients).

To reveal the effect of rotation cropping and bacterial manure on the growth of Chrysanthemum morifolium and screen the beneficial endophytic, the diversity of endophytic and dominant genera of different treatment groups were analyzed. Four different treatments were continuous cropping, rotation, self-made organic fertilizer and commercially available fertilizer, respectively. Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results showed that enzyme Hae III was more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae III was more than that of enzyme Hinfl. In comparison of diversity, the endophytic bacterial communities' diversity index in group of cropping rotation and fertilizer was higher than that of continuous cropping which indicated that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte. 18 dominant species were selected, including 3 kinds of Firmicutes, 4 kinds of Actinomycetes and 11 kinds of Proteobacteria. The results of dominant species comparison showed that the number of dominant species in continuous cropping of Ch. morifolium was significantly less than that of the rotation group. Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter, Streptomyces, Streptomyces, Flavobacterium and Mycobacterium were not found in the continuous cropping of Ch. mortfolium group. Dominant species of fertilizer treatment group was similar with the rotation group, and the continuous cropping group's dominant species was more abundant. It indicates that these bacteria may be able to mitigate hindrance in continuous cropping, especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention. Compared with leaves, there are more dominant species in roots and stems. The diversity of edophytic bacterial communities in continuous cropping of Ch. morifolium stays below than that in the rotation of Ch. morifolium, and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.
Actinobacteria ; physiology ; Agriculture ; Biodiversity ; Chrysanthemum ; growth & development ; microbiology ; Deoxyribonucleases, Type II Site-Specific ; Endophytes ; Fertilizers ; Gram-Positive Bacteria ; physiology ; Phylogeny ; Plant Leaves ; Plant Roots ; microbiology ; Polymorphism, Restriction Fragment Length ; Proteobacteria ; physiology ; RNA, Ribosomal, 16S ; chemistry ; genetics ; Soil ; Soil Microbiology

OBJECTIVETo investigate the expression of fatty acid synthase (FAS) in adenosis, atypical ductal epithelial hyperplasia, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of breast, and the correlation of FAS expression with HER2 gene amplification in IDC.

METHODSImmunohistochemical EnVision method staining for FAS was performed in 100 cases of breast lesions and 10 normal breast tissues. HER2 gene amplification was detected with FISH in 60 cases of IDC.

RESULTSThe cohort included 10 cases of adenosis, 10 atypical ductal epithelial hyperplasia, 20 DCIS (8 high-grade, 9 intermediated-grade and 3 low-grade), and 60 cases of IDC (5 grade 1, 40 grade 2 and 15 grade 3). FAS expression was negative in all 10 normal breast tissues; in the 10 cases of adenosis, strongly positive FAS expression was detected in one case, positive in 2, weakly positive in 4, and negative in 3; in the 10 cases of atypical ductal epithelial hyperplasia, FAS immunohistochemistry showed that 1 was strongly positive, 4 positive, 4 weakly positive, and 1 negative; in the 20 cases of DCIS, FAS immunostaining showed that 12 were strongly positive, 5 positive, 1 weakly positive, and 2 negative; FAS expression showed a clear increasing trend from normal breast tissue, atypical ductal epithelial hyperplasia to DCIS (χ(2) = 42.02, P < 0.01). Likewise, the increasing trend was also demonstrated from adenosis to DCIS (χ(2) = 34.69, P < 0.01). There was also a positive correlation between FAS expression and extent of lesion among normal breast tissue, adenosis, atypical ductal epithelial hyperplasia and DCIS (χ(2) = 86.02, P < 0.01; r = 0.568, P < 0.01). FAS expression was not correlated with the grade of DCIS (χ(2) = 9.12, P = 0.16). In the five cases of grade 1 IDC, FAS immunostaining showed that 4 cases were strongly positive and 1 positive; in the 40 cases of grade 2 IDC, FAS immunostaining showed that 27 strongly positive, 12 positive, and 1 negative; in the 15 cases of grade 3 IDC, FAS immunostaining showed that 6 were strongly positive, 5 positive, 3 weakly positive, and 1 negative; FAS expression was stronger and more extensive in DCIS, IDC grades 1 and 2 than that in other groups. However, FAS expression was weaker in the IDC grade 3 (χ(2) = 11.26, P = 0.01). The positive expression rate of FAS in IDC was generally higher than that in benign breast lesions (χ(2) = 47.19, P < 0.01). In the 60 cases of IDC, FISH showed HER2 gene amplification in 22 cases, but not in the remaining 38 cases. FAS expression in IDC was highly correlated with HER2 gene amplification (r = 0.44, P < 0.01). The expression of FAS had significant correlation with status of ER and PR and tumor size (P < 0.05). There was no significant correlation with age, immunohistochemical HER2 expression, lymph node metastasis and clinical stage (P > 0.05).

CONCLUSIONSFAS may be closely related to the carcinogenesis of breast IDC. FAS expression is closely associated with HER2 gene amplification in IDC.

Breast ; metabolism ; pathology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Fatty Acid Synthases ; metabolism ; Female ; Fibrocystic Breast Disease ; metabolism ; Gene Amplification ; Genes, erbB-2 ; Humans ; Hyperplasia ; Lymphatic Metastasis ; Middle Aged ; Receptor, ErbB-2 ; metabolism

OBJECTIVETo evaluate the clinical effectiveness of three localization methods, including methylene blue, metal clips and intraoperative colonoscopy in laparoscopic colorectal surgery.

METHODSA retrospective analysis was performed to review the clinical data of 64 patients who underwent the laparoscopic colorectal operations in Cancer Hospital of Fudan University from December 2009 to June 2012. Three methods of tumor localization were used perioperatively, including 23 cases of methylene blue, 20 of metal clips and 21 of colonoscopy.

RESULTSOperations were successfully performed in this cohort and there were no deaths or complications. In methylene blue group, intraoperative colonoscopy was performed in two cases because of the inability to visualize blue dye on the serosal surface of the intestinal wall, another 2 cases were converted to open operation because of methylene blue diffusion and inability to identify resection margin. Intraoperative colonoscopic localization was required for 3 cases of sigmoid colon or upper rectal tumor because of inaccurate tumor localization by metal clips. Poor operative exposure due to obvious bowel distension prompted the conversion to open surgery in 2 cases of colonoscopy localization group, and the accurate position of the lesion was not found in another 2 cases due to long pedunculated adenoma.

CONCLUSIONSColorectal tumor can be localized effectively by endoscopic methylene blue tattooing at a maximum of 2 tumors before operation and the method of 4-point positioning can significantly improve the accuracy of colorectal tumor localization. Tumor localization preoperatively on the day of surgery by metal clip is accurate for the right or left colon cancer. Intraoperative colonoscopy can localize tumor accurately and rapidly for rectosigmoid or descending tumor, and the incidence of bowel distension can be significantly reduced. Localization method should be considered according to the tumor location and surgical procedure.

Adult ; Aged ; Colorectal Neoplasms ; surgery ; Female ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo screen and analyze CD34(+) cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression.

METHODSCD34(+) cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs, miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity.

RESULTSOf the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34(+) cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant (P < 0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34(+) cells from the bone marrow of AML patients compared with the CD34(+) cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34(+) cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34(+) cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c.

CONCLUSIONA variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34(+) cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34(+) cells from the bone marrow of AML patients.

Antigens, CD34 ; metabolism ; Female ; Hematopoietic Stem Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Oligonucleotide Array Sequence Analysis

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.
Antigens, CD ; genetics ; immunology ; Antigens, CD34 ; genetics ; immunology ; Antigens, Differentiation, Myelomonocytic ; genetics ; immunology ; Aptamers, Nucleotide ; metabolism ; Biomarkers ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; genetics ; immunology ; Nucleic Acid Conformation ; SELEX Aptamer Technique ; Sialic Acid Binding Ig-like Lectin 3