OBJECTIVE:To investigate the differences of the active components of different parts of rhubarb(the head,the body and the end part of the root)from genuine medicinal materials in Gansu province so as to provide theoretical basis for its quality control.METHODS:A comparative study of the fingerprints of different parts of rhubarb extract were conducted by HPLC,in which,the C18 was used as the chromatographic column under room temperature,the mobile phase consisted of menthol-0.1%H3PO4(70∶30)with a flow rate of 1.0ml/min,the column's temperature of room temperature and detection wavel_ ength of 280nm.RESULTS:Significant differences were noted in the HPLC fingerprints of different parts of rhubarb,there were significant differences in the contents of the active components such as emodin,chrysophanol,etc.,the contents decreased progressively from the head of root,the body part to the end part.CONCLUSION:In order to ensure the medical value and the quality of rhubarb medicinal material,different parts of which should be differentiated in the quality control.

Objective Multimeric cyclic RGD (Arg-Gly-Asp) peptides are capable of improving the integrin αvβ3-binding affinity due to the polyvalence effect.In this study,the authors prepare 99Tcm-la-bearing cyclic RGD tetramer E{E[c(RGDfK)]2}2,and evaluate its biodistribution and imaging in nude mice beating U87 MG human glioma xenografts with integrinαvβ3-positive.Methods 99Tcm-hydrazino-nictinamide (HYNIC)-E{E[c(RGDfK)]2}2 was prepared by two-step method,while HYNIC wag chosen as bifunctional chelator,and tricine and trisodium triphenylphosphine-3,3,3-trisuifonate (TPPTS) as coligands.The af-finity of c (RGDyK) monomer,HYNIC-E[c(RGDfK)]2 dimer and HYNIC-E{E[c(RGDfK)]2}2 tetramer to integrin αvβ3 was compared by in vitro competitive assay against binding of 125I-c(RGDyK)to integrin αvβ3.positive U87 MG human glioma cells.The biodistribution [the percentage of injection dose per gram of tissue(%ID/g)] and imaging were performed in nude mice bearing UB7MG human glioma xenografts.Re-suits The labeling yield of 99Tcm-HYNIC-E{E[c(RGDfK)2}2 was over 95%,and the radiochemical purity was more than 99%after purification with Sop-Pak C18 cartridge.The 50%inhibiting concentration (IC30) val-ues of c(RGDyk),HYNIC-E[c(RGDfK)]2 and HYNIC-E{E[c(RGDfK)]2}2 were 85.9,9.5 and 4.5 nmol/L, respectively.The result indicated that RGD tetramer possessed a significantly higher affinity to in-tegrinαvβ3.The biodistribution data showed that 99Tcm-HYNIC-E{E[c(RGDfK)]2}2 was excreted mainly through kidneys.The tumor uptake of 99Tcm-HYNIC-E{E[c(RGDfK)]2}2 was two times higher than 99Tcm- HYNIC-E[c(RGDfK)]2,at 1h postinjection,with the uptake of(10.32±0.07)%ID/g and(5.15±0.52)%ID/g,respectively,which was consistent with the in vitro competitive binding data.The tumor up-tale of 99Tcm-HYNIC.E{E[c(RGDfK)]2}2 was still as higher as(9.35±1.35)%ID/g at 4 h postinjec-tion, which demonstrated that the retention time of radiotracer in tumor was long enough.The imaging showed that tumor was clearly visualized at 1h postinjection,and the image at 4 h postinjection Was better.Conclusion The higher tumor uptake and longer retention time in tumor make 99Tem-HYNIC-E{E[c(RG-DfK)J 2}2 a promising radiotracer for integrinαvβ3-positive tumors imaging,furthermore,suggest that radi-onuelides(such as 90Y).1abeled RGD tetramer is more suitable for the therapy of integrin αvβ3-positive tumors.

Objective To explore the application of hydrogen proton magnetic resonance spectroscopy (1H-MRS) in the diagnosis of peripheral tumor cell infiltration of gliomas. Methods Forty patients with glioma were examined by 1H-MRS preoperation, and were divided into low grade glioma group (n=20) and high grade glioma group (n=20) according to postoperative pathological diagnosis. Tumor resection with peripheral tissues marked previously was carried out under the guidance of neuronavigator system. All the pathological sections were divided into positive group and negative group according to the presence or absence of tumor cells, and the differences in pathological findings of peripheral regions (region 1, 2 and 3) and 1H-MRS values were analyzed in these two groups. Results No infiltration was found in the peripheral regions of low grade glioma group except for one case in peripheral region 1, while infiltration was found in all peripheral regions of high grade glioma group. There was no significant difference in 1H-MRS values between positive group (n=24) and negative group (n=36) in patients with high grade glioma (P>0.05). Conclusion 1H-MRS enjoys some advantages over routine radiological examinations in the diagnosis of peripheral tumor cell infiltration of gliomas. Total removal can be expected when combined with neuronavigator system, while there is room for improvement for relevant techniques.

OBJECTIVETo observe the expression of FLI-1 in primitive neuroectodermal tumors (PNET), explore the value of immunohistochemical staining of FLI-1 in combination with other neural markers in diagnosis of PNET, and analyze the prognostic factors in PNET patients.

METHODS35 cases of PNET, of which 33 cases with complete clinical data, were included in this study. Immmunohistochemistry (The En Vision method) was applied to detect the expression of FLI-1, CD99, Syn, NSE, S-100, NF, Vim in the tumor tissues. The clinicopathological data of 33 cases were analyzed by Cox regression.

RESULTSThe positive expression rate of FLI-1 were 51.4% and that of CD99 was 88.6%. The sensitivity of FLI-1 combined with CD99 was up to 100%. The positive rates of Vim, Syn, NSE, s-100 and NF were 91.4%, 48.6%, 45.7%, 22.9% and 0, respectively. Cox regression analysis showed that the impact of primary location and treatment modality were of statistical significance (P < 0.05), but the age, sex, stage or size of tumors did not (P > 0.05).

CONCLUSIONImmunohistochemical detection of FLI-1 and neural markers is a preferred method for clinical diagnosis of PNET. The main factors affecting the prognosis are the primary location of PNET and treatment modality.

12E7 Antigen ; Adolescent ; Adult ; Antigens, CD ; metabolism ; Brain Neoplasms ; metabolism ; pathology ; therapy ; Cell Adhesion Molecules ; metabolism ; Child ; Child, Preschool ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Infant ; Male ; Middle Aged ; Neuroectodermal Tumors, Primitive ; metabolism ; pathology ; therapy ; Neuroectodermal Tumors, Primitive, Peripheral ; metabolism ; pathology ; therapy ; Pelvic Neoplasms ; metabolism ; pathology ; therapy ; Phosphopyruvate Hydratase ; metabolism ; Proportional Hazards Models ; Proto-Oncogene Protein c-fli-1 ; metabolism ; S100 Proteins ; metabolism ; Survival Rate ; Synaptophysin ; metabolism ; Vimentin ; metabolism ; Young Adult

OBJECTIVETo detect the immunoreactivity of targeted fusion anti-caries DNA vaccine pGJA-P in vitro, and the ability to enhance the immune responses compared with the non-targeted fusion anti-caries DNA vaccine pGLUA-P.

METHODSThe CHO cells were transfected with pGJA-P and the expression of recombinant protein in cultured supernatants were detected using Western blotting. 5 to 6-month-old female Japanese rabbits were immunized with either pGJA-P or pGLUA-P via either intramuscular injection (i.m.) or intranasal route (i.n.). The sera and saliva were collected and the antibody responses were checked by ELISA. The effect of immune sera on the synthesis of water-insoluble glucan by glucosyltransferase of S. mutans was examined.

RESULTSThe expressed protein could response to specific anti-GTF antibody. The antibody responses in serum generated by pGJA-P via i.m. were significantly higher than those generated by pGLUA-P (P < 0.01). The antibody responses in saliva generated by pGJA-P via i.n. were significantly higher than those generated by pGLUA-P (P < 0.01). The higher mucosal antibody response induced by pGJA-P via i.m. compared with pGLUA-P (P < 0.01) was detected. The immune sera of rabbits immunized by pGJA-P via i.m. most significantly inhibited the synthesis of water-insoluble glucan by glucosyltransferase.

CONCLUSIONSThe recombinant protein expressed by pGJA-P had the immunoreactivity to anti-GTF antibody. pGJA-P could induce faster and higher specific mucosal SIgA antibody responses via i.n. or serum IgG antibody responses via i.m. compared with non-targeted DNA vaccine, pGLUA-P. High titres of specific mucosal antibodies were found in rabbits immunized with pGJA-P via i.m. The immune sera of rabbits immunized by pGJA-P via i.m. displayed the ability of inhibiting the synthesis of water-insoluble glucan by glucosyltransferase.

Animals ; Bacterial Proteins ; immunology ; CHO Cells ; Cricetinae ; Dental Caries ; prevention & control ; Female ; Glucosyltransferases ; immunology ; metabolism ; Immunoglobulin G ; blood ; Membrane Glycoproteins ; immunology ; Rabbits ; Recombinant Fusion Proteins ; administration & dosage ; immunology ; Streptococcus mutans ; immunology ; Transfection ; Vaccines, DNA ; administration & dosage ; immunology

OBJECTIVETo investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression.

METHODSAffymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data.

RESULTSCompared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression.

CONCLUSIONSHepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.

Carcinoma, Transitional Cell ; genetics ; pathology ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cyclin D1 ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; physiology ; Humans ; Nuclear Proteins ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Proteins ; genetics ; physiology ; Urinary Bladder Neoplasms ; genetics ; pathology

OBJECTIVETo investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells.

METHODSThe A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants.

RESULTSSpecific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX.

CONCLUSIONSCTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

Animals ; Antigens, CD ; genetics ; immunology ; metabolism ; CTLA-4 Antigen ; Cricetinae ; Dental Caries ; prevention & control ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo use a glycemic method to screen hepatocellular carcinoma (HCC) metastasis related aberrant 1-6 fucosylated glycoproteins, and to analyze the metastasis-related alterations of annexin II.

METHODS2-DE coupled with lectin affinity blot, lectin affinity precipitation followed by MALDI-TOF-MS/MS was established to screen glycoproteins related to HCC metastasis. Immunofluorescence analysis, Western blot and real-time PCR were performed on higher and lower metastatic HCC cell lines to detect the protein expression levels and mRNA levels of annexin II.

RESULTSThe Lens culinaris agglutinin (LCA) affinity glycoprotein profiles from MHCC97-L and MHCC97-H cells were differentially displayed when compared with Hep3B. Annexin II was identified by MALDI-TOF-MS/MS and its increased core-fucosylation was associated with HCC metastasis and it was confirmed. In addition, we found that annexin II was distributed in the cytoplasm and it had higher protein and gene expressions in MHCC97-L and MHCC97-H cells than in Hep3B cells.

CONCLUSIONOur results suggest that the increase of annexin II and its expression levels, and the increase of core-fucosylation might all be related to HCC metastatic ability.

Annexin A2 ; analysis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression ; Humans ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; genetics

Objective (1) To evaluate the effect of insertion of two 15-amino-4,7,10,13-tetraoxapentadecanoic (2 PEG4 ) linkers into cyclic Arg-Gly-Asp (RGD) Dimer E [c(RGDfK)]2 on receptor binding in vitro, (2) to assess its biodistribution in vivo and (3) to investigate the value of 99Tcm labeled 2PEG4-Dimer for integrin αvβ3-positive tumors imaging.Methods The expression of U87 human glioma cells and integrin αv β3 was determined by immunofluorescence staining.The half-inhibition concentrations (IC50) for 125 I-cyclo (Arg-Gly-Asp-D-Tyr-Lys) (c(RGDyK) ) of c ( RGDyK ), hydrazinonictinamide ( HYNIC )-Dimer and HYNIC-2PEG4-Dimer binding to integrin αvβ3 were measured.99Tcm-HYNIC-Dimer and 99Tcm-HYNIC-2PEG4-Dimer were synthesized using non-SnCl2 formulation.Biodistribution and imaging studies were performed in nude mice bearing human glioma xenografts.The unpaired t test was used for statistical analysis.Results The labeling yield of the two radiotracers was more than 95%, and the radiochemical purity was more than 99% through Sep-Pek C18 cartridge.HYNIC-2PEG4-Dimer had significantly higher binding affinity of integrin αvβ3 than c(RGDyK) and HYNIC-Dimer (IC50 = 0.8 nmol/L, 27 nmol/L and 2.4 nmol/L, respectively).Biodistribution study showed that 99Tcm-HYNIC-2PEG4-Dimer was mainly excreted via the kidney.The tumor uptake of 99Tcm-HYNIC-2PEG4-Dimer was higher than that of 99Tcm-HYNIC-Dimer at 2h post injection ((5.71 ±0.96) and (2.10 ±0.50) % ID/g, t =4.80, P＜0.05).The xenografted tumors were visible at 0.5 h post injection and the image contrast increased with time due to the tracer clearance of the background tissue.Conclusion 99 Tcm-HYNIC-2PEG4-Dimer is a promising radiotracer for integrin αvβ3-positive tumor imaging.

The dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine. Bellamya purificata is widely distributed in the Chang Jiang River basin, it is natural diets of W. pigra. Current study was conducted to compare and analyze the nutritional ingredient in W. pigra, body fluid and flesh of B. purificata. Results showed that the contents of protein, crude fat and total sugar in W. pigra, body fluid and flesh of B. purificata were significantly different (P < 0.05). Protein content in W. pigra accounts up to 65.01%. The contents of inorganic elements and amino acid were abundant in W. pigra, body fluid and flesh of B. purificata. The content of essential amino acids in them were 32.6, 221.59, 40.78 mg x g(-1), respectively. The content of flavor amino acid in them were 27.51, 14.5, 32.03 mg x g(-1), while the coresponding content of antioxidant amino acid were 8.81, 5.91, 9.73 mg x g(-1), respectively. The individual amino acids of high content in them were Glu, Asp and Leu. Macro elements Ca, P, Mg and trace elements Zn, Si, Fe were abundant. It could be speculated that W. pigra may be a promising novel food, and the present results provide a foundation to develop artificial feed for W. Pigra.
Amino Acids ; analysis ; Animals ; Gastropoda ; chemistry ; Leeches ; chemistry ; Medicine, Chinese Traditional