The aim of this study was to investigate the expression of matrix metalloproteinase 26 (MMP-26), tissue inhibitor of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase 9 (MMP-9) in patients with diffuse large B cell lymphoma (DLBCL) and their correlations with pathogenesis and development of DLBCL. A total of 95 specimens excised from DLBCL patients were prepared. Expression of MMP-26, TIMP-4 and MMP-9 were tested by SABC immunohistochemistry method and its correlation to clinicopathology indexes were analyzed. The results showed that as compared with reactive hyperplasia of lymph nodes, the high expression of MMP-26, TIMP-4 and MMP-9 were found in different types of DLBCL. The positive expression rate of MMP-26 was related to immune typing (P < 0.05). The expression level of MMP-26 in GCB was lower than that in non-GCB, and did not relate to clinical staging, age, sex, diseased region (P > 0.05). The positive expression rate of MMP-9 was related to clinical staging, the positive expression rate of MMP-9 proteins in patient at III and IV stage was obviously higher than that in patients at I and II stage, but did not relate to immune type, age, sex and diseased region of DLBCL (P > 0.05). The expression of TIMP-4 did not relate to immune type, clinical stage, age, sex, disease region (P > 0.05). The expression of MMP-26 in pathologic tissue of DLBCL did not relate to expression of TIMP-4, but positively related to expression of MMP-9 protein (r = 0.486, P < 0.05). It is concluded that MMP-26 and MMP-9 synergically express in DLBCL. MMP-26 may be involve in pathogenesis and invasiveness of DLBCL, the expression of MMP-26 relates to subtypes of DLBCL. The MMP-26 may serve as an indicator for typing of DLBCL and contributes to predict the invasion and metastasis of DLBCL and itself may become a potential target for therapy.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Humans ; Lymphoma, Large B-Cell, Diffuse ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Matrix Metalloproteinases, Secreted ; metabolism ; Middle Aged ; Tissue Inhibitor of Metalloproteinases ; metabolism ; Young Adult

To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.
Animals ; Cell Line ; Cells, Cultured ; Chickens ; Dependovirus ; genetics ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

AIMTo identify the main metabolites of oxymatrine (OMT) in rats.

METHODSTo optimize the conditions of LC/ESI-ITMS' chromatograms and spectra by oxymatrine and matrine (MT), and summarize their ionization and cleavage rules in ESIMS, then serving as the basis for the metabolite analyses of oxymatrine in rats. To collect the 0-24 h urine samples of the rats after ip 40 mg x kg(-1) oxymatrine, the samples were enriched and purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC/ESI-ITMS. The structures of OMT metabolites were identified according to their retention times and ESI-ITMSn rules.

RESULTSSix phase I metabolites and the parent drug OMT were found in the rat urine, and the main metabolite was MT. No phase II metabolites were found.

CONCLUSIONThe developed LC/ESI-ITMSn methods to identify the metabolites of oxymatrine in rats is not only simple and rapid but also sensitive and specific. This technology is one of the most efficient methods for the analysis of drug metabolites.

Alkaloids ; isolation & purification ; pharmacokinetics ; urine ; Animals ; Chromatography, Liquid ; methods ; Plants, Medicinal ; chemistry ; Quinolizines ; isolation & purification ; pharmacokinetics ; urine ; Rats ; Rats, Wistar ; Sophora ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods

AIMTo estabilish a rapid and sensitive LC-ESI-ITMSn method for the identification of ephedrine and its main metabolites in rat urine.

METHODSAfter optimizing the detection condition of LC-ESI-ITMSn chromatography and mass spectrometry by using a standard ephedrine, the ionization and cleavage rules of ephedrine in ESI-MS and ESI-MSn modes were summarized, and then serving as the basis for the metabolite analysis of ephedrine in rat urine. Rat urine samples of 0-48 h were collected after ig 10 mg x kg(-1) ephedrine, then the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC-ESI-ITMSn.

RESULTSThe structures of ephedrine metabolites were elucidated according to the changes of the molecular weights of the metabolites (deltaM) and their cleavage pattern in ESI-ITMSn. As a result, three phase I metabolites and the parent drug ephedrine were identified existing in rat urine, but no phase II metabolites were found.

CONCLUSIONThe LC-ESI-ITMSn method is rapid and highly sensitive and sepecific, it is suitable for the identification of ephedrine and its metabolites in rat urine.

Animals ; Chromatography, High Pressure Liquid ; methods ; Ephedrine ; chemistry ; metabolism ; urine ; Male ; Molecular Weight ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods

AIMTo establish a LC-MS(n) method for the identification of anisodamine and its metabolites in rat feces.

METHODSFeces samples were collected after single administration of 25 mg x kg(-1) anisodamine to rats, and dipped in water for 1 h. Samples were then extracted by ethyl acetate. The pretreated samples were separated on a reversed-phase C18 column using a mobile phase of methanol / 0.01% triethylamine (adjusted to pH 3.5 with formic acid) (60 : 40, v/v) and detected by LC-MS". Identification of the metabolites and elucidation of their structures were performed by comparing their changes in molecular masses (deltaM), retention-times and full scan MS(n) spectra with those of the parent drug and blank feces.

RESULTSThe parent drug and its seven metabolites (6beta-hydroxytropine, nor-6beta-hydroxytropine, aponoranisodamine, apoanisodamine, noranisodamine and hydroxyanisodamine, tropic acid) were found in rat feces.

CONCLUSIONThis method is sensitive, rapid, simple, effective, and suitable for the rapid identification of drug and its metabolites in biologic samples.

Animals ; Feces ; chemistry ; Rats ; Rats, Wistar ; Solanaceous Alkaloids ; analysis ; metabolism ; Tandem Mass Spectrometry ; methods

AIMTo identify anisodine and its metabolites in rat plasma after ingestion of anisodine by combining liquid chromatography and tandem mass spectrometry (LC-MS(n)).

METHODSPlasma samples from rats after a single orally administration of 20 mg anisodine were added with methanol to precipitate protein. Then, it was analyzed by LC-MS(n). Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses, retention-times and full scan MS(n) spectra with those of the parent drug and blank plasma.

RESULTSThe results revealed that the parent drug and its four metabolites (norscopine, scopine, hydroxyanisodine, N-oxide anisodine) existed in rat plasma.

CONCLUSIONThis method is sensitive, rapid, simple, and it is suitable for the rapid identification of drug and its metabolits.

Administration, Oral ; Animals ; Chromatography, Liquid ; methods ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Scopolamine Derivatives ; isolation & purification ; metabolism ; Sensitivity and Specificity ; Solanaceae ; chemistry ; Tandem Mass Spectrometry ; methods

AIMTo identify the main metabolites of stachydrine in rat.

METHODSThe ionization, cleavage and chromatographic characteristics of stachydrine were studied by using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometry (HPLC-ESI/MS) for the first time. These characteristics of stachydrine were used as the basis for the analyses of metabolites in rat urine. The 0 - 24 h urine samples of rats after ig 25 mg x kg(-1) stachydrine were collected and purified by using C10 solid-phase extraction cartridge, and then analyzed by HPLC-ESI/MS to identify stachydrine and its metabolites.

RESULTSThe parent drug (stachydrine), 6 phase I metabolites (N-demethyl, dehydrogenation, ring-oxidation) and 2 phase II metabolites (glycine conjugates of 2 ring-oxidation products) were identified existing in rat urine.

CONCLUSIONThe presented method was proved to be sensitive, rapid, high selective and specific for the identification of stachydrine and its metabolites in rat urine.

Animals ; Chromatography, High Pressure Liquid ; methods ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Proline ; analogs & derivatives ; isolation & purification ; metabolism ; urine ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Stachys ; chemistry

AIMTo establish a rapid and sensitive LC-MSn method for the identification of trigonelline and its main metabolites in rat urine.

METHODSAfter optimizing the detection conditions of LC-MSn chromatography and mass spectrometry using trigonelline, its ionization and cleavage in ESI-MS and ESI-MSn modes were summarized, then serving as the basis for the metabolite analysis of trigonelline in rat urine. The 0-48 h urine samples of rats were collected after iv 8 mg x kg(-1) trigonelline, then, the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC-MSn.

RESULTSThe structures of trigonelline metabolites were elucidated according to the changes of the molecular weights of the metabolites (deltaM) and their cleavage pattern in ESI-ITMSn. As a result, two phase I metabolites and the parent drug were identified existing in rat urine, and two phase II metabolites were identified.

CONCLUSIONThe LC-MSn method is rapid and high sensitive and specific, it is suitable for the identification of trigonelline and its metabolites in rat urine.

Alkaloids ; chemistry ; isolation & purification ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; methods ; Hypoglycemic Agents ; chemistry ; isolation & purification ; metabolism ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Trigonella ; chemistry

AIMTo identify the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine.

METHODSUrine samples from 0 - 24 h were collected after single ig dose of 500 mg x kg(-1) daidzein to each of six rats. The urine samples were purified by SPE column (SPE C18) and analyzed with liquid chromatographic-tandem electrospray ionization ion trap mass spectrometry (LC-ESI/MS(n)) for potential metabolites.

RESULTSSeveral new hydroxylate metabolites and its sulfate conjugates were found and identified in rat urine.

CONCLUSIONLC-ESI/MS(n) is proved to be a simple, rapid, sensitive and specific technique for identification of the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine.

Animals ; Chromatography, Liquid ; methods ; Hydroxylation ; Isoflavones ; chemistry ; metabolism ; urine ; Male ; Molecular Structure ; Phytoestrogens ; chemistry ; metabolism ; urine ; Rats ; Rats, Sprague-Dawley ; Reproducibility of Results ; Seeds ; chemistry ; Soybeans ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Sulfates ; metabolism ; Tandem Mass Spectrometry ; methods

AIMTo identify the main metabolites of jatrorrhizine in rat urine.

METHODSThe rat urine samples were collected 0 - 72 h after ig 12 mg x kg(-1) jatrorrhizine, then the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by combining liquid chromatography and tandem electrospray ionization ion trap mass spectrometry (LC-ESI/ITMS(n)). Identification and structural elucidation of the metabolites were performed by comparing the changes in molecular masses, retention-times and full scan MS(n) spectra with those of the parent drug.

RESULTSAt least seven phase I metabolites (such as de-methyl, de-hydrogen and hydroxyl metabolites) and eleven phase II metabolites (such as glucuronide conjugates and methyl-conjugates) were identified in rat urine.

CONCLUSIONThe developed LC-ESI/ITMS(n) method is not only simple and rapid but also sensitive and specific for the identification of metabolites of jatrorrhizine in rat urine.

Animals ; Berberine ; analogs & derivatives ; isolation & purification ; metabolism ; urine ; Chromatography, High Pressure Liquid ; methods ; Coptis ; chemistry ; Molecular Structure ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Spectrometry, Mass, Electrospray Ionization ; methods