ABSTRACT:OBJECTIVE The aim of this study was to identify Alisma orientale (Sam.)Juzep.and its adulterants using the ITS2 barcode.METHODS The total DNAs were extracted from twenty-eight samples of A.orientale as well as adulterants. The ITS2 sequences of these samples were amplified and sequenced.The acquired sequences were submitted to the GenBank and the other ITS2 sequences of 17 samples from ten species were downloaded from the GenBank.Total 45 ITS2 sequences were aligned and the genetic distances among them were analyzed with MEGA 5.1.The sequences analyses were performed u-sing the BLAST1 and the nearest distance methods,and were presented intuitively by constructing Neighbor-joining(NJ)tree. RESULTS The lengths of all ITS2 sequences of A.orientale and A.plantago-aquatica were 311 bp.Only one mutation exis-ted among them.There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 se-quences.The NJ tree showed that A.orientale is obviously different from its adulterants,which showed high monophyly. CONCLUSION As a DNA barcode,ITS2 sequences can stably and accurately distinguish A.orientale from its adulterants and also provide a new clue to identify the medical germplasm resources and ensure the clinical safety in utilization of Chinese mate-ria medica.

OBJECTIVE: To investigate the prevalence rate, malocclusion type and treatment rate as well as awareness of malocclusion among children and adolescents living in the Hangzhou municipality. METHODS: 1818 children and adolescents ages 7 approximate, equals 16 living in Hangzhou municipality were evaluated. RESULTS: The overall malocclusion rate was 35.75%. In Angle's classification: Angle I 593 cases (32.62%), Angle II 48 cases(2.64%),Angle III 9 cases (0.49%). Among the malocclusion type of crowding was mixed dentition 70.66%,permanent dentition 85.89%. In the overjet malocclusion mixed dentition was noted in 60.57% and permanent dentition in 51.05%.The type of overbite was mixed dentition 67.82%,permanent dentition 31.23%. In the crossbite of anterior teeth mixed dention was noted in 12.30% and permanent dention in 9.91%. Overall treatment rate for malocclusion was 10.15%.CONCLUSION: Among Hangzhou municipality juveniles there is both inadequate prevention and treatment of dental malocclusion.

OBJECTIVETo construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown.

METHODSWe chose the binding domain of Raf-1, PI3K, RalGDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double enzyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression efficiency of the plasmids in eukaryotic cells was detected by Western blot analysis.

RESULTSFive of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T cells, and (RBD+CRD)(Raf-1)- U-Box-pcDNA3.1 can knocked down the protein level of Ras in PANC-1 cells.

CONCLUSIONSWe successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.

Cloning, Molecular ; Genetic Vectors ; HEK293 Cells ; Humans ; Phosphatidylinositol 3-Kinases ; genetics ; Plasmids ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Ubiquitin-Protein Ligases ; genetics ; ral Guanine Nucleotide Exchange Factor ; genetics ; ras Proteins ; genetics

BACKGROUNDIt is important to identify the multiple sites of leptin activity in obese women with breast cancer. In this study, we examined the effect of exogenous human leptin on heat shock protein 70 (HSP70) expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.

METHODSWe cultured MCF-7 human breast cancer cells and established nude mice bearing xenografts of these cells, and randomly divided them into experimental and control groups. The experimental group was treated with human leptin, while the control group was treated with the same volume of normal saline. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues. Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells. Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.

RESULTSLeptin activated HSP70 in a dose-dependent manner in vitro: leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells (P < 0.001). There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group (P > 0.05). Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group (P > 0.05).

CONCLUSIONSA nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor. HSP70 may be target of leptin in breast cancer. Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.

Animals ; Blotting, Western ; Breast Neoplasms ; drug therapy ; metabolism ; Cell Line, Tumor ; Female ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Leptin ; pharmacology ; therapeutic use ; Mice ; Mice, Nude ; Real-Time Polymerase Chain Reaction ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effect of Compound Qingqin Liquid (CQL) on the expression level of angiotensin II (Ang II) and COX-2 mRNA transcription and protein expression in the renal tissue of rats with uric acid nephropathy.

METHODSSD rats were randomly divided into the blank control group, the model group, the positive drug group, the high, moderate, and low dose CQL group according to number randomization principle. The model was established by gastrogavage of adenine, accompanied with yeast feeding. Distilled water was given by gastrogavage to rats in the blank control group and the model group. Allopurinol at the daily dose of 9.33 mg/kg was given by gastrogavage to rats of the positive control group. CQL at the daily dose of 3.77 g/kg, 1.89 g/kg, and 0.09 g/kg was respectively given by gastrogavage to rats in the high, moderate, and low dose CQL groups. All treatment lasted for 6 weeks. Rats were randomly divided at week 4 (3 in the blank control group, and 6 in the rest groups), and the rest rats were killed at week 6. The renal tissue was extracted. The expression level of Ang II and COX-2 mRNA transcription were detected by RT-PCR. The expression level of Ang II was detected by ELISA. The expression level of COX-2 protein was detected by Western blot and immunohistochemical assay.

RESULTSCompared with the blank control group, except the mRNA expression of Ang II at week 4, the mRNA and protein expression of Ang II and COX-2 obviously increased at week 4 and 6 in the model group (P < 0.01, P < 0.05). The COX-2 protein expression at week 4 was obviously lower in the high and moderate dose CQL groups than in the model group and the low dose CQL group (P < 0.05); the average integral of optical density value was obviously lower in the positive control group than in the model group. Except the mRNA expression of Ang II in the high dose CQL group at week 6, the mRNA and protein expression of Ang II obviously decreased in the positive control group and each dose CQL group (P < 0.01, P < 0.05). Of them, the effects were better in the high and moderate dose CQL groups than in the positive control group and the low dose CQL group (P < 0.05, P < 0.01). Besides, the mRNA expression of COX-2, the average integral of optical density value were obviously lower in the positive control group and each dose CQL group than in the model group (P < 0.05). The protein expression of COX-2 was obviously lower in the high and moderate dose CQL groups than in the model group (P < 0.05). Of them, the mRNA expression of COX-2 was better in the moderate dose CQL group than in the positive control group (P < 0.05); the protein expression of COX-2 was better in the high dose CQL group than in the low dose CQL group (P < 0.05).

CONCLUSIONCQL was capable of lowering the expression level of Ang II, COX-2 mRNA transcription and protein expression, thus suppressing the inflammatory pathological injury of the renal tissue.

Angiotensin II ; metabolism ; Animals ; Cyclooxygenase 2 ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; metabolism ; Kidney Diseases ; drug therapy ; metabolism ; Male ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Uric Acid

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

Objective To understand the status and influential factors of those neglect of left-behind children in rural area,and to provide bases for the development of intervention measures.Methods 2917 students were selected as the study subjects from Changfeng county of Anhui province with cluster sampling method and were evaluated by a Parents-Child Conflict Tactics Scales and questionnaire on influential factors.Results 1694 left-behind children,accounted for 58.1％ of the total students,were surveyed in this investigation.The prevalence rates of neglect,among total children,left-behind children,non-left-behind children were 67.4％,70.2％,63.5％,respectively.The prevalence of neglect among left-behind children was higher than that among non-left-behind children (x2=14.322,P＜0.000).There were no significant associations with the neglect rate of left-behind children regarding gender or age differences.Result from multivariate logistic regression analysis indicated that the neglect among the left-behind children were associated with family dysfunction(OR values of moderate and serious family dysfunctions compared to good family function were 1.628 and 2.341,respectively)and the rate of keeping in touch with parents(OR values of sometimes and seldom keeping in touch compared to regular in touch were 1.299 and 1.844,respectively).The starting age of being left-behind(OR values of starting age that being left-behind from 6 to 10 and ≤5 years relative to starting age of left-behind ≥11 years were 0.703 and 0.630,respectively)appeared to be the protection factor to the neglect of those left-behind children.Conclusion Our findings indicated that the status of neglect among the left-behind children was serious.Prevention programs on the issue should target on a number of factors,including the characteristics of the chldren them-selves,as well as on the family of the children.

OBJECTIVETo examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC).

METHODSOVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR.

RESULTSThe CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.

CONCLUSIONThese data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.

Animals ; CD40 Ligand ; genetics ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Cytokines ; secretion ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; metabolism ; Female ; Interleukin-12 ; secretion ; Interleukin-23 ; secretion ; Interleukins ; secretion ; Mice ; Ovarian Neoplasms ; metabolism ; pathology ; Th1 Cells ; secretion ; Transfection

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics

BACKGROUNDIt is essential to clarify the interactions of hormones during the progression of human breast cancer. This study examined the effects of exogenous human leptin on estrogen receptor (ER) alpha and beta in human breast tumor tissue in a nude mouse xenograft model.

METHODSWe created nude mice xenografts of MCF-7 human breast cancer cells, and randomly divided them into an experimental group and a control group. The mice in experimental group were injected subcutaneously around tumors with human leptin, while the control group were injected with the same dose of normal saline. A real-time RT-PCR assay was developed to quantify the mRNA of ERalpha, beta in the tumor tissues. Western blotting analyses were used to assess the relative quantities of the ERalpha, beta proteins.

RESULTSLeptin-treated xenografted nude mice were successfully established. The amount of ERalpha mRNA was significantly higher in the leptin group than in the control group (P < 0.01), while the amount of ERbeta mRNA was significantly lower in the leptin group than in the control group (P < 0.01). Western blotting analyses revealed that the ERalpha protein level was significantly higher in the leptin group than in the control group (P < 0.01), while the ERbeta protein level was significantly lower in the leptin group than in the control group (P < 0.01).

CONCLUSIONSNude mouse xenograft model can be safely and serviceably treated with human leptin by subcutaneous injections around tumor. ERalpha, beta were both targets of leptin in breast cancer. Leptin can up-regulate the expression of ERalpha and down-regulate the expression of the ERbeta in human breast tumor.

Animals ; Blotting, Western ; Breast Neoplasms ; drug therapy ; genetics ; metabolism ; Cell Line, Tumor ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; Leptin ; therapeutic use ; Mice ; Mice, Nude ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; Random Allocation ; Xenograft Model Antitumor Assays