Objective　To explore the correlation between structure domains and function of chemokine receptor CXCR4. Methods　After the establishment of wild type chemokine receptor CXCR4 and CXCR2, 5 CXCR4/CXCR2 chimeras, 2 CXCR4 mutants stably expressed on CHO cell line, all variants' bound activity with the ligant recombinant human SDF-1β，signal transduction ability after stimulation and their function as coreceptor for HIV-1 were studied with ligand-binding assay, cytosensor/microphysiometry and cell-cell receptor gene fusion assay. Results　Among all 7 changed CXCR4 receptors, 3 chimeras (2444a, 4442, 4222) and 1 mutant (CXCR4-Tr) could be bound with SDF-1β in various degrees, of which only 2444a totally and CXCR4-Tr partially maintained signaling. All changed receptors but 4222 could act as coreceptors for HIV-1 (LA1) in varying degrees. Conclusion　Several structure domains of CXCR4 are involved in the binding with SDF-1β. Among these domains, N-terminal extracellular domain has high affinity bound capability with SDF-1β, and the 3rd extracellular loop contributes to the binding too. Although the C-terminal intracellular domain has no association with the maintenance of the overall structure of the receptor and the ligand bound capability, the signaling is decreased when this domain is truncated. For CXCR4 signaling, not only the conserved motif DRY box is needed, but also the characterized conformation of the whole molecule must be formed when activation is required. There are some overlaps between SDF-1β bound domains and coreceptor funtion domain in molecular structure of CXCR4.

OBJECTIV E To optimize the s alt-processing technology of Rosa laevigata ，and to study high performance liquid chromatography（HPLC）fingerprints and chromaticity values of R. laevigata before and after salt-processing. METHODS The comprehensive scoring method was adopted to optimize the salt-processing technology of R. laevigata using appearance character ， moisture and polysaccharide content as index. Fingerprints were established by HPLC method before and after salt-processing ，and chromaticity values （L*，a*，b*）of the powder before and after salt-processing were determined. The multivariate statistical analysis was carried out for raw product and salt-processing product of R. laevigata by using common peak areas and chromaticity values as index. RESULTS The optimal salt-processing technology of R. laevigata was to mix it with appropriate amount of salt water ，place them in the preheated frying wok at 140 ℃，fry them for 25 min，and rotate frying wok 20 times/min. Ten common peaks were calibrated by HPLC fingerprints before and after salt-processing ，and 3 components were identified ，such as gallic acid ，catechin and ellagic acid. The chromaticity values L*，b* and E* changed significantly after salt-processing. The multivariate statistical analysis method could distinguish raw products and salt-processing products into two categories ，in which peaks 1，5，6 and 10 and chromaticity values b* and E* were important characteristic factors. CONCLUSIONS The optimized salt-processing technology is stable and reliable ，and the established fingerprint has good repeatability and stability. Fingerprint and chromaticity values combined with multivariate statistical analysis can provide reference for the identification and quality analysis of R. laevigata before and after salt-processing.

OBJECTIVE To establish the fingerprints of dried Houttuynia cordata and its decoction pieces ，conduct chemometrics analysis and determine the contents of 5 flavonoids such as neochlorogenic acid. METHODS High performance liquid chromatography （HPLC）method was adopted. Using quercitrin as reference ，HPLC fingerprints of 10 batches of dried H. cordata and its decoction pieces were drawn. The similarity evaluation was conducted by Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition），the common peaks were also confirmed. SIMCA-P 14.1 software was applied for principal component analysis （PCA）and partial least square-discriminant analysis （PLS-DA），and the variable importance in projection（VIP）value more than 1 was considered as a standard to screen the differential components affecting the quality of these two products ；meanwhile，the contents of 5 components such as neochlorogenic acid in both products were determined by the same HPLC method. RESULTS There were 20 common peaks in 10 batches of dried H. cordata and 10 batches of its decoction pieces with the similarity values more than 0.960. A total of 5 common peaks were identified ，which were neochlorogenic acid （peak 1）， chlorogenic acid （peak 3），cryptochlorogenic acid （peak 4），rutin（peak 7）and quercitrin （peak 11）. The results of PCA and PLS-DA showed that dried H. cordata could be distinguished from its decoction pieces obviously ；the common peaks with VIP value greater than 1 were as follows ：peak 7（rutin），peak 20，peak 5，peak 13，peak 2，peak 18，peak 3（chlorogenic acid ）， peak 14，peak 17 and peak 19. The linear range of neochlorogenic acid ，chlorogenic acid ，cryptochlorogenic acid ，rutin and quercitrin were 3.77-60.29 μg/mL（r＝0.999 7），1.40-22.42 μg/mL（r＝0.999 5），3.76-60.22 μg/mL（r＝0.999 9），2.19-35.06 μg/mL （r＝0.999 9）and 25.49-407.88 μg/mL（r＝0.999 7），respectively. RSDs of precision ，stability（24 h）and reproducibility E-mail：20190394@njucm.edu.cn tests were all lower than 3％. The average recoveries of the above components in these two products were 98.72％-101.12％ and 98.86％ -100.63％ with RSDs less than 3％（n＝9）. In dried H. cordata ，the average contents of 5 components were 0.87，0.33，0.59，0.61 and 6.17 mg/g，while the average contents were 0.42，0.11，0.26，0.23 and 3.16 mg/g in its decoction pieces ，respectively. CONCLUSIONS HPLC fingerprint and the method of content determination are stable and feasible ，which could be used for the quality control of dried H. cordata and its decoction pieces. Besides ，rutin and other components may be the differential components which could affect the quality of these two products ；the average contents of the 5 flavonoids such as neochlorogenic acid in dried H. cordata all decrease after processing.

Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Infant ; Male ; Prognosis ; Retrospective Studies ; Treatment Outcome ; Vesico-Ureteral Reflux ; diagnosis ; therapy

OBJECTIVETo investigate arsenic trioxide (As(2)O(3))-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML).

METHODSHepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 micro mol/L As(2)O(3) for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML.

RESULTSThe growth rates of HepG2 cells were slower in the As(2)O(3) treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As(2)O(3) treated groups. The expression of PML decreased in HepG2 cells with 2 micro mol/L As(2)O(3) treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 micro mol/L As(2)O(3), and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 micro mol/L As(2)O(3).

CONCLUSIONSOur results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As(2)O(3) induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As(2)O(3) may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As(2)O(3) treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As(2)O(3) therapy.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Humans ; Neoplasm Proteins ; metabolism ; Nuclear Matrix ; drug effects ; metabolism ; Nuclear Proteins ; Oxides ; pharmacology ; Promyelocytic Leukemia Protein ; Transcription Factors ; metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Proteins

It is unclear why the concentration of testosterone increases in the testicular vein after hemicastration without corresponding alteration in gonadotropins. The present work was undertaken to examine whether the testosterone levels could be modified by denervation of the testis in adult rats. Both systemic and testicular blood samples were collected either immediately before or 6 and 24 hours after hemicastration from the rats two weeks after denervation of either inferior spermatic nerves (ISN) or ISN plus superior spermatic nerves (ISN-SSN). Increase of testicular testosterone induced by hemicastration was significantly (P<0.05) inhibited in these rats, as compared with the sham animals (at 6 and 24 hours, ISN vs sham: 16.00±3.35 vs 42.72±13.85 and 26.93±8.68 vs 71.16±13.30 whilst ISN-SSN vs sham: 31.63±7.92 vs 60.61±18.11 and 27.70±8.93 vs 93.92±19.73 ng/ml, respectively), whereas no significant change in LH was observed in all the experimental groups. FSH underwent no alteration in all the ISN denervation groups, but a significant elevation was observed in the ISN-SSN denervation groups (P<0.05) before hemicastration. Therefore, it appears that the change in FSH is not the cause of the inhibition of testosterone increase in the hemicastrated rats after testicular denervation and that ISN plays an active role in regulation of testosterone increase induced by hemicastration.

OBJECTIVETo investigate the pro-angiogenic effects of several multiple myeloma (MM) cell line culture supernatants on human bone marrow endothelial cell (HBMEC) proliferation, migration, and capillary formation, and the anti-angiogenic effects of thalidomide.

METHODSHBMEC was cultured in the presence of MM cell lines (IM9, XG1, U266 and MOLP-5) supernatants. Proliferation and migration of HBMEC were determined, capillary-like tubule formation of HBMEC was examined in fibrin and Matrigel. The inhibiting effect of thalidomide was investigated by adding it into myeloma cell line culture supernatants. Vascular endothelial growth factor (VEGF) was measured by ELISA.

RESULTS(1) MM cell lines culture supernatants promoted HBMEC proliferation and migration. (2) In fibrin and Matrigel, capillary-like tubule network formation promoted by the supernatants. (3) All of these effects could be inhibited by thalidomide. (4) This effect was not related to VEGF in the supernatants.

CONCLUSIONSMM cell line promote proliferation, migration and tubule formation by secreting VEGF or other several cytokines. Thalidomide can inhibit these effects.

Angiogenesis Inhibitors ; pharmacology ; Bone Marrow ; blood supply ; Cell Division ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Endothelial Growth Factors ; metabolism ; Endothelium, Vascular ; cytology ; drug effects ; physiology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Lymphokines ; metabolism ; Multiple Myeloma ; pathology ; secretion ; Neovascularization, Physiologic ; drug effects ; Thalidomide ; pharmacology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate diagnostic values of the detection of TMPRSS2-ERG gene fusion in metastatic prostate cancer.

METHODSA total of 32 fine needle aspiration (FNA) specimens of metastatic prostate carcinomas were retrieved from the pathology files at MD Anderson Cancer Center. The metastatic sites included the pelvic and remote lymph nodes, liver, bone, and thyroid gland. Immunohistochemical staining for PSA, PAP, synaptophysin, chromogranin A was performed. TMPRSS2-ERG gene fusion was evaluated on sections of cell blocks by fluorescence in situ hybridization (FISH) using ERG gene break-apart probes.

RESULTSThe mean age of the patients was 67 years. Twenty-six patients had a previous history of prostatic adenocarcinoma, while 6 patients presented initially with metastasis. In 11 patients, the metastatic lesions showed characteristic features of small cell carcinoma (SCC) and were positive for synaptophysin (9/9), chromogranin A (7/8), but negative for prostatic specific antigen (7/7). FISH analysis demonstrated a rearrangement of ERG gene in 10 of 32 cases (31.3%), and the rearrangement was associated with deletion of the 5' ERG gene in 6 cases. In addition, the copy number of ERG rearrangement gene locus was increased in 8 cases. Among the 11 cases with SCC features, a rearrangement of ERG gene was present in 5 cases, of which a deletion of the 5' ERG gene and increased copy number were seen in 3 cases.

CONCLUSIONSTMPRSS2-ERG gene fusion can be evaluated in FNA specimens of metastatic prostate cancer. Metastatic prostate cancers have a high prevalence of TMPRSS2-ERG gene fusion along with a frequent copy number increase of ERG gene. TMPRSS2-ERG gene fusion persists in metastatic prostate cancers and even in those with poorly differentiated SCC features. Therefore, an identification of the TMPRSS2-ERG gene fusion may be used to establish the prostatic origin of metastasis.

Acid Phosphatase ; Adenocarcinoma ; genetics ; metabolism ; pathology ; secondary ; surgery ; Aged ; Aged, 80 and over ; Biopsy, Fine-Needle ; Carcinoma, Small Cell ; genetics ; metabolism ; pathology ; secondary ; surgery ; Chromogranin A ; metabolism ; Follow-Up Studies ; Gene Fusion ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Liver Neoplasms ; genetics ; metabolism ; pathology ; secondary ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Protein Tyrosine Phosphatases ; metabolism ; Synaptophysin ; metabolism

Myeloperoxidase (MPO) is released from activated neutrophils.The inflammation in preeclampsia was found to be associated with endothelial dysfunction.We hypothesized that cardiac and circulating MPO levels are elevated in hypertensive pregnancy.Systolic and diastolic blood pressure and heart rate were measured on pregnancy days 14,16,18 and 20 in normal pregnant and hypertensive pregnant rats.Left and right ventricle weights,the number of viable fetuses,litter size,fetal and placenta weights were recorded on gestational day 21.Circulating and cardiac MPO activities,soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor (VEGF) and nitric oxide (NO) were detected.The results showed increases in cardiac (left,but not right ventricle) and circulating MPO activities,and concomitantly lower number of viable fetuses,litter size,and fetal and placenta weights,and decreases in NO in hypertensive pregnant rats.Also,the increases in circulating sFlt-1 and VEGF were found in hypertensive pregnant group.In conclusion,maternal and fetal detrimental changes along with increases in circulating sFlt-1 and VEGF in hypertensive pregnancy may be associated with increases in cardiac and circulating MPO activities,confirming the causative role of inflammatory response in preeclampsia.

OBJECTIVETo investigate the effects of mono(2-ethylhexyl) phthalate(MEHP), the primary metabolite of di(2-ethylhexyl) phthalate (DEHP), on testosterone biosynthesis in Leydig cells cultured from the Sprague Dawley rat testis.

METHODSBased on the primary Leydig cell culture model, MEHP exposure groups involved control (0 micromol/L), 62.5, 125, 250, 500 and 1000 micromol/L. We observed mitochondria activity with the MTT method, measured the testosterone level with RIA and determined steroidogenesis acute regulatory protein (StAR) mRNA expression with RT-PCR.

RESULTSAfter Leydig cells were exposed to MEHP for 24 hours, the activity of mitochondria enhanced evidently at 250 micromol/L and then declined markedly at 1000 micromol/L compared with the control group (P < 0.01). The testosterone level showed an increasing tendency in both basal and hCG-stimulated states with statistical significance at 250 and 500 micromol/L compared with the control group (P < 0.01). However, the expression of StAR mRNA appeared unchanged at 62.5, 125 or 250 micromol/L, but exhibited a decreasing tendency at 500 and 1000 micromol/L (P < 0.01).

CONCLUSIONME- HP directly affected the activity of mitochondria and testosterone biosynthesis of the Leydig cells in vitro. StAR, the regulator of cholesterol transport into mitochondria, might not be responsible for the increase of testosterone biosynthesis induced by MEHP.

Animals ; Cells, Cultured ; Diethylhexyl Phthalate ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Leydig Cells ; drug effects ; metabolism ; Male ; Phosphoproteins ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; biosynthesis