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Objective. To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. Methods. After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. Results. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. Conclusion. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα. [ABSTRACT FROM AUTHOR]

Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 °C. When there is sufficient Na+ in the medium (≥25 mM), [3H]2β-carbomethoxy-3β-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 °C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, d-amphetamine, p-tyramine, and dl-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 °C. [Copyright &y& Elsevier]

The combined thermo gravimetric-infrared-mass spectrometry (TG-IR-MS) analysis were conducted under the temperature of 30° to 1 100° with a heating rate of 10°/min to monitor the different rank coal's pyrolysis and nitrogen release behavior online.The thermal analysis results show that the various nitrides like HCN and NH3 are released from coals during pyrolyzing.The released products vary with different coalification coals during pyrolysis.Nitrogen is mainly released in the forms of NH3 and HCN in gas coal,1/3 coking coal and coking coal, but NH3 in the high rank coal.The HCN and NH3 come from different types of nitrogen in the coal's internal structure.The HCN may be mainly derived from the edge of five-membered rings pyrrole nitrogen and six-membered rings pyridine nitrogen in coals, the NH3 from the inside quartery nitrogen.The release of NH3 experience two stages:the lower temperature stage at which it is from the elementary pyrolysis of volatile components;and the high temperature stage at which it is from the secondary pyrolysis of volatile components. [ABSTRACT FROM AUTHOR]

In this study, taking Cistanche deserticola in Xinjiang as the experimental material, the optimal process for extracting polysaccharides from C. deserticola with water extraction was studied by using single factor and orthogonal experiment. Its effects on protein removal and polysaccharides retaining were investigated by using Sevag, enzymatic method or combination of these two methods, so as to determine the optimal method for protein removal from polysaccharides of C. deserticola; the decolorization and purification methods such as macroporous resin of AB-8 and activated Carbon were used to determine the optimal process. The results showed that the extraction rate of polysaccharides from C. deserticola was 18.40% during the optimal process of the water extraction as follows: extraction temperature 75 ℃, extraction time 165 min and solid-liquid ratio 1∶55. The protein removal rate can reach 31.40% and polysaccharide retention rate can reach 96.00% under the optimal protein removal process: temperature 50 ℃, time 2 h, and papain dosage 0.2%. The decolorization rate of activated Carbon and macroporous resin called AB-8 was 80.37% and 86.43%, and the recovery rate of polysaccharides was 77.05% and 91.93%, respectively, suggesting that macroporous resin was more suitable for decoloration. Macroporous resin named AB-8 increased the purity of the polysaccharide crude extract from 67.70% to 84.80% under the following conditions: concentration of the sample 4 g·L~(-1), concentration of the eluent 60% ethanol, and the flow rate 1 mL·min~(-1), showing significant purification effect.

Aim: To investigate the possible correlation between osteoglycin expression and gelatinase activity of mouse hepatocarcinoma Hca-F cells., Methods: A eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was constructed and transfected into Hca-F cells to investigate the possible correlation between osteoglycin expression and gelatinase activity of Hca-F cells cultured with extract of lymph node, liver, spleen or in DMEM medium. The activity of gelatinases was examined through zymographic analysis., Results: High expression of osteoglycin attenuated the gelatinase activity of Hca-F cells cultured with extract of lymph node, and at the same time, decreased the metastatic potential of Hca-F cells to peripheral lymph nodes in vivo., Conclusion: High expression of osteoglycin decreases the gelatinase activity of Hca-F cells cultured with extract of lymph node; regulation of gelatinase activity might be one of mechanisms that osteoglycin contributes to lymphatic metastasis suppression.

Background & Objective: Metastasis is a complex process involving multiple genetic changes. The high mortality and poor prognosis caused by metastasis in malignant tumor patients and the uncertain mechanisms are always the prominent problems in the field of oncology. In order to screen for lymphatic metastasis-associated genes, the gene expression profiles of mouse hepatocarcinoma cell lines Hca-F (highly metastatic) and Hca-P (low metastatic) were compared by gene chip., Methods: Total RNA was isolated from Hca-F and Hca-P cells, and synthesized into double-stranded cDNA, then synthesized into biotin-labeled cRNA probes by in vitro transcription. The cRNA probes were separately hybridized with Affymetrix GeneChip MOE430A (containing 22,690 transcripts, including 14,500 known mouse genes and 4,371 ESTs), and the signals were scanned by the GeneArray Scanner. The results were analyzed by bioinformatics., Results: Compared with the gene expression profile of Hca-P cells, 901 (6.2%) genes and 129 (3%) ESTs were up-regulated by at least 2 folds in Hca-F cells; 33 genes, including endoglin (EDG; CD105), Mcam (Muc18; Mel-CAM; CD146), Cdc42ep5 (CEP5; Borg3), Ptprr (protein tyrosine phosphatase, receptor type, R), F2r [coagulation factor II (thrombin) receptor; Par1; ThrR], D7Ertd458e (necl-5), NR1D1, Serpin h1 (HSP47), AXL, Mak, and Areg (AR), were up-regulated 13.93-29.86 folds. According to Gene Ontology and Treeview analysis, these 33 genes were involved in angiogenesis, cell adhesion, signal transduction, cell motility, transcription, chaperone activity, protein kinase activity, receptor binding, and so on., Conclusion: Many lymphatic metastasis-associated genes were screened by high-throughput gene chip method; validating their cellular functions will help to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.

Aim: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential., Methods: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip(r) MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics., Results: Out of the 14 500 known genes investigated, 110 (0.8%) were up regulated at least 2(3) fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 2(3) fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile., Conclusion: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

Objective: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis., Methods: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed., Results: Ten differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones., Conclusion: SSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.

Aim: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential., Methods: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing., Results: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes., Conclusion: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 degrees C. When there is sufficient Na+ in the medium (> or =25 mM), [3H]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 degrees C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, D-amphetamine, p-tyramine, and DL-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 degrees C.

Background & Objective: Metastasis is the leading cause of tumor-related death, in which lymphogenous metastasis is the most common pattern and also a bridgehead of further metastasis. However, the molecular mechanism of metastasis is uncertain. This study was designed to investigate the correlation between the metastasis of hepatocarcinoma cell(HCC) to lymphnode and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymphnodes., Methods: The maps of MMPs in supernatants of mouse HCCs with different metastatic potential Hca-F(high potential) and Hca-P cells(low potential) cultured with extract of lymph node, liver or spleen were examined by zymographic analysis. Growth fraction of lymphocytes in lymph nodes of tumor burden mice was detected by flow cytometry. The apoptosis signals of macrophages in lymph nodes were observed with TUNEL staining. The expressions of Fas ligand of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry., Results: With the presence of the extraction of lymph note, the quantity of MMP-9 significantly increased (P < 0.01): in RPMI1640 medium, the quantity of MMP-9 produced by Hca-F and Hca-P were 1256 +/- 157 and 2642 +/- 385, respectively. After the addition of extraction of lymph node, the quantity of MMP-9 increased to 12,403 +/- 894 and 9086 +/- 686, respectively, together with the secretion of a large amount of MMP-2 (Hca-F: 7364 +/- 2001, Hca-P: 2997 +/- 1990) and active MMP-9 (Hca-F: 7297 +/- 1657, Hca-P: 3914 +/- 1253), and the amount of which was also more in Hca-F than in Hca-P cells(P < 0.05). There was no MMPs detected when the extraction of liver and spleen is present. The growth fraction of lymphocytes was as follow: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post-inoculation and then decreased rapidly, while in the Hca-P cells, the peak appeared on the 7th day post-inoculation and then kept at a high level. TUNEL staining showed that the positive signals of macrophages were detected around Hca-F cells. The expression of Fas ligand of Hca-F cells was stronger than that of Hca-P cells (P < 0.01)., Conclusion: The MMPs secretion of Hca-F and Hca-P cell depend on the environment of lymph nodes. The increased expression of Fas ligand protein in Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.