Pyrolysis polygeneration of pine nut shell: Quality of pyrolysis products and study on the preparation of activated carbon from biochar.

A lab-scale pyrolysis reactor was utilized to investigate the effect of pyrolysis temperature (300-700°C) on the yield, quality, and energy distribution of products issued from the pyrolysis polygeneration of pine nut shells. Afterward, activated carbon was prepared from biochar using the steam activation method. Pyrolysis temperatures ranging from 500 to 600°C were found to be optimal in inducing products with improved properties, such as higher heating values of non-condensable gas, lower water content and elevated heating values of bio-oil, and substantial fixed carbon content and greater specific surface area of biochar. In addition, it was noticed that the activation conditions had a significant effect on the yield and adsorption performance of the activated carbon. As a result, activated carbon with elevated specific surface area reaching 1057.8m(2)/g was obtained at the optimal conditions of 850°C activation temperature, 80min activation time, and 1.5 steam/biochar ratio.

Geldanamycin, a heat shock protein 90-binding agent, disrupts Stat5 activation in IL-2-stimulated cells.

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone in eukaryotic cells. Hsp90 plays a critical role in regulating signal transduction pathways that control cell proliferation since its chaperone function is restricted to a subset of proteins including some signal molecules. Improper function of these proteins can be induced by an anti-tumor agent geldanamycin (GA) which is the specific inhibitor of Hsp90. In this study, it was demonstrated that GA interferes with IL-2-stimulated proliferation of murine CTLL-2 cells. As to the signaling mechanisms underlying this inhibitory effect, we discovered GA disrupts the IL-2-stimulated activation and phosphorylation of the transcription factor Stat5, indicating the proper function of Hsp90 is indispensable for Stat5 activation. This conclusion is validated by the observation that Hsp90 interacts with Stat5 in the immunoprecipitation assay and GA interrupts their interaction. Furthermore, by constructing deletion mutants, we identified the c-terminal half of Stat5 coiled-coil region is responsible for binding with Hsp90.

Long term gene therapy of Parkinson's disease using immortalized rat glial cell line with tyrosine hydroxylase gene.

Glial cell is an ideal vehicle for gene therapy of brain diseases. However, there are many limits in using primary glial cells. Therefore, an immortalized rat glial cell line (RGLT) was established by SV40 large T-antigen (LTag) gene from the primary rat fetal glial cells. The RGLT cell was shown to be non-tumorigenic after transplantation to nude mice (up to 4 weeks) and rat striatum (up to 18 months). Rat tyrosine hydroxylase (TH) gene was transfected into RGLT cell to obtain RGLT-TH cell. The TH immunohistochemical staining and HPLC-ECD analysis demonstrated the TH expression and dopamine (DA) production in RGLT-TH cells in vitro. When implanting RGLT-TH cells into the striatum of 6-hydroxydopamine (6-OHDA) lesioned hemiparkinsonism model rats, TH immunohistochemical staining showed the TH presence in striatum and HPLC-ECD analysis held at 6 months after cell implantation showed an increase of DA content in striatum. The asymmetric rotation of rats receiving RGLT-TH cells was reduced by 50%-60% and this reduction persisted stably at least for 18 months. These results suggest that the immortalized glial cell line could serve as an ideal vehicle for therapeutic gene delivery system to achieve a long-term gene therapy of neurodegenerative diseases.

Ref-1 protein enhances the IL-2-stimulated telomerase activity.

Telomerase is an important ribonucleoprotein enzyme involved in cellular proliferation and senescence. Activation of telomerase has been detected in a vast majority of human cancer cells. In this article, we demonstrated that Interleukin-2 (IL-2) which is the pivotal cytokine in the immune system could stimulate the activity of telomerase in the cultured BA/F3beta cells. It was also found that the level of IL-2-induced telomerase activity was decreased by the treatment with chemical oxidant in vitro. Since IL-2 stimulation produces a oxidative shift of the intracellular environment, the activation and maintenance of telomerase in this oxidative circumstance requires particular protection. Here we proved the redox factor-1 (Ref-1) protein was involved in this process. The addition of GST-Ref-1 protein increased the level of IL-2-induced telomerase activity in the TRAP assay, while elimination of the endogenous Ref-1 protein by immunodepletion decreased it. Consistent with these in vitro results, IL-2-induced telomerase activity could be enhanced by transient overexpression of Ref-1 protein in BA/F3beta cells. Taken together, these findings proved that Ref-1 protein benefits the activation of telomerase activity in the oxidative microenvironment of the BA/F3beta cells stimulated by IL-2.

[Anti-tumor effect through human endostatin gene transfer in mice bearing B16 melanoma].

OBJECTIVE: To investigate the effects of direct intratumor injection of the packaged cells with retroviral vector carrying human endostatin (hEN) on the growth inhibition of B16 melanoma in C57/BL6 mice. METHODS: Retroviral vector, pLNC-hEN, was constructed with modified and identified hEN gene. The cell line, PA317, was used to establish ecotropic virus producing cells by transfecting and packing with pLNC-hEN. Then the cells were injected directly into the tumor in C57/BL6 mice bearing B16 melanoma, established by intra-cutaneous injection of B16 cell suspension. The tumor size was measured at different intervals to observe the antitumor effect. Micro-vessel density (MVD) in the tumor tissue was evaluated by immunohistological examination to count the apoptotic cells by TUMEL staining. RESULTS: Tumor with diameter of 2 - 3 mm was observed in all mice after 7 - 9 days. The average tumor volume on D3, D5, D7 and D9 after gene transfection was 4.67 +/- 1.1, 22.25 +/- 13.06, 84.17 +/- 43.5 and 155.08 +/- 81.1 mm(3) in the gene therapy group but 136.17 +/- 30.61, 390.17 +/- 220.47, 1 021.67 +/- 537.4 and 2 920.2 +/- 220.01 mm(3) in the control group, the difference of which was statistically significant. The average MVD in the gene therapy and control groups were 8 +/- 2.28 and 28.17 +/- 5.31 while the average apoptotic cell number in the two groups were 23.33 +/- 3.83 and 2.33 +/- 1.21, both of which were statistically significant. CONCLUSION: The direct injection of packaged cells carrying hEN gene is able to inhibit the growth of micro-blood vessels and promote tumor cell apoptosis, which ultimately inhibits the growth of B16 melanoma.

[Therapeutic effects of human interleukin 10 gene transfer on severe acute pancreatitis in rats, an experimental study].

OBJECTIVE: To study the therapeutic effects of human interleukin 10 (IL-10) gene transfer on severe acute pancreatitis (SAP) in rats. METHODS: Twenty healthy SD rats were injected intraperitoneally with SA liposome, SA liposome/pcDNA3 or SA liposome/pcDNA3-IL-10. Another twenty SD rats were randomly divided into five groups: rats in one group underwent laparotomy only (normal control), and SAP was induced in the other 4 groups induced by homogeneous injection of sodium taurocholate beneath the pancreatic capsule. Among the 4 SAP groups, one group did not receive any drugs, and liposomes, pcDNA3 or pcDNA3-IL-10 complexed with cationic liposomes were administered to the other groups. Drugs were administered by a single intraperitoneal injection thirty minutes after SAP had been induced. The levels of IL-10 in pancreas, liver and lungs were determined by ELISA kits. The level of serum amylase, histology, and tissue tumor necrosis factor (TNF) were assessed and mortality rate was observed in different groups for one week. RESULTS: The levels of IL-10 in the pancreas, liver and lung 24 hours after IL-10 gene transfer, increased significantly (all > 350 pg/g), and then gradually decreased, however, the levels of IL-10 were still significantly higher that those in the control groups (P < 0.05) 96 hours later and decreased to normal in one week. The levels of IL-10 of transfer control group were not significantly different from those of the normal control group. The levels of IL-10 expression in pancreas, liver and lungs were increased significantly in the gene therapy group, compared with the SAP group. The serum amylase level was (4 300 +/- 700) U/L in normal control group, increased to (20 300 +/- 1 100) U/L 24 hour after SAP induction without a difference between the therapy control group and SAP group, and decreased to (6 800 +/- 700) U/L after IL-10 gene therapy (P < 0.05). The histological score of pancreas was 4.1 +/- 0.2 24 hours after the induction of SAP, and was 3.2 +/- 0.3 in the IL-10 therapy group. The level of TNF in pancreas, liver, and lungs 24 hours after the induction of SAP was significantly higher than that in normal control group (P < 0.05) and was not different from that in therapeutic control group. However, it was decreased markedly in IL-10 therapy group (P < 0.05). No rat in any group died within 2 days after onset. There was no difference of mortality between SAP group and therapeutic control group. The one-week mortality was 90% in the whole SAP group. The one-week mortality of IL-10 gene therapy group was 30 %, significantly lower than that in SAP group (P < 0.05). There was no significant difference in the therapeutic control groups and the SAP group. The values of relative risk of SAP group, SA liposome group, and pcDNA3 group were 12, 8, and 11 times higher than that of gene therapy group (P < 0.05). CONCLUSION: Cationic liposome mediated pcDNA3-IL-10 gene therapy decreases significantly the severity and mortality of SAP.

Further identification of NSF* as an epilepsy related gene.

Previous data proved that NSF* was an epilepsy related gene (ERG1). In this study, using phosphorothioate oligodeoxynucleotide (PS-ODN), an antisense of NSF to downregulate the function of NSF in vitro cultured hippocampus neurons and PC12, this treatment simultaneously induced enhancement of the neurite outgrowth of hippocampal neurons and PC12, a phenomenon similar to the structural changes following epilepsy. Immunocytochemistry analysis showed that the enhancement of neurite outgrowth was in a sequence-specific manner and Northern blot confirmed that the decrease of NSF mRNA levels in PC12 was in a dose-dependent manner. Moreover the expression of NSF was downregulated during differentiation of PC12 induced by NGF and high KCl. Therefore, providing more evidence to support the fact that NSF was an ERG1.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats.

BACKGROUND: Studies have proven the validity of interleukin-10 (IL-10) in the treatment of experimental pancreatitis. Prophylactic human IL-10 (hIL-10) gene treatment attenuated the severity in cerulein models. Our research aims to study whether the therapeutic hIL-10 gene could decrease both severity and mortality in a lethal pancreatic model. METHODS: Severe acute pancreatitis (SAP) was induced by sodium taurocholate. A plasmid-hIL-10 construct (pcDNA3-hIL-10) complexed with cationic liposomes was administered to SAP rats by a single intraperitoneal injection. Levels of hIL-10 in the pancreas, liver, and lungs were determined by ELISA kits. The severity of pancreatitis was assessed in terms of serum amylase, histology, and tissue tumor necrosis factor alpha (TNF-alpha). Mortality, observed for 7 days, was evaluated for gene therapy or control groups. RESULTS: After hIL-10 gene therapy, hIL-10 levels in the pancreas, liver, and lungs increased significantly and the serum amylase, tissue TNF-alpha, and histological changes in pancreas, liver, and lungs decreased markedly. Therefore, mortality was significantly reduced in the hIL-10 gene therapy group, in which 70% of rats survived in the 7-day observation, while only 10% survived in untreated groups (P < 0.05). CONCLUSION: We found that liposome/hIL-10 gene therapy decreased severity and mortality in SAP, even carried out after SAP establishment, predicting a more convenient shift to clinical applications.

Treatment of Parkinson's Disease by Genetically Modified Immortalized Fibroblasts.

Primary rat fibroblast cells were immortalized by genetic modification of SV40 large T antigen (LT(Ag)) gene and called RFLT. This cell line was non-tumorigenic after grafting into nud-mouse and rat. The LT(Ag)gene stopped expression when the cells were transplanted in rat striatum, but it resumed the expression ability after the transplanted cells were recovered from striatum and cultured in vitro.TH gene and GCH gene were transfected into RFLT, respectively, and two types of transfected cells, RFLT-TH and RFLT-GCH, were obtained. In mixed culture with these two cell lines, DA was detected with HPLC-ECD. Implanting mixture of those cells into the striatum of PD rats significantly decreased their rotational asymmetry for up to 12 weeks. The expression of TH gene was proved by TH immunohistochemical staining in the sections of rat brain. The establishment of the genetically modified immortalized cells may play role in the gene therapy of PD.

Transcription Regulation of bcl-2 Gene.

Various extracelluar stimulation, including those by growth factors and cytokines, can induce the bcl-2 gene expression. Bcl-2 protein induced by this stimulation seems to be essential for cell survival. In order to understand the regulations of bcl-2 transcription, recent advances at transcriptional and post-transcriptional levels in this field will be described.

Interaction between Jak3 and Nucleosome Assembly Protein 1.

Tyrosine kinase Jak3 plays a critical role in the interleukin 2 IL-2 signaling because it not only participates the Jak-Stat pathway, but also interacts with unidentified signal transducers and regulates expression of some oncogenes such as c-fos and c-myc. Abundant evidence demonstrated that phosphorylated tyrosine was necessary for the interaction between two proteins. Therefore, in order to clarify the role of Jak3 in IL-2 signal transduction, the tyrosine-phosphorylation-involved yeast two-hybrid system was constructed and the N-terminal region JH3-JH7 of Jak3 was used as a bait to screen a peripheral blood cDNA library. About 50 double-positive colonies were obtained. Sequence analysis indicated that one of them was from nucleosome assembly protein 1 gene (Nap1), and encoded a protein of 392 amino acid residues. Two-hybrid system results demonstrated that interaction between Jak3 and Nap1 depended on the level of tyrosine phosphorylation. Furthermore, immunoprecipitation and Western blot experiments confirmed that Jak3 really interacted with Nap1 in murine pro-B lymphocyte BAF/BO3beta cells.

Ubiquitin Conjugating Enzyme Ubc9 is Involved in Protein Degradation of Redox Factor-1 (Ref-1).

Redox factor-1 (Ref-1) is a bifunctional protein playing an important role in both cellular redox regulation and DNA apurinic/apyrimidinic sites' repair. To find Ref-1interacting proteins (Rips), a yeast two-hybrid screening was performed by using Ref-1 redox domain as the 'bait', and five positive clones were obtained. One of them (Rip3) was identified to be the ubiquitin-conjugating enzyme Ubc9. Simultaneous overexpression of Ubc9 in Hela cells dramatically inhibited the enhancement of AP-1 reporter gene by Ref-1. Western blot indicated that the protein level of Ref-1 dropped down as the result of simultaneous overexpression of Ubc9. These results suggest that Ubc9 is involved in the protein degradation of Ref-1, resulting in the downregulation of Ref-1 physiological function.

A Novel Type of Apoptosis Induced by Cadmium in BA/F3beta Cells.

Apoptosis is usually accompanied by DNA fragmentation and up-regulation of reactive oxygen species, and it can be inhibited by overexpression of Bcl-2. Here, cadmium was found to induce apoptosis in BA/F3beta cells. MTT assay, Hochest 33258 staining, and transmission electron microscopy analysis were used to detect the apoptosis, however, neither DNA fragmentation nor up-regulation of reactive oxygen species were observed in this type of apoptosis. Furthermore, Bcl-2 overexpression had no effect on this type of apoptosis. In conclusion, these data suggested that cadmium induced a novel type of apoptosis in BA/F3beta cells.

Adenovirus-Mediated th Gene Therapy of Parkinson's Disease in a Experimental Rat Model.

A recombinant adenovirus (AdCMV th) encoding tyrosine hydroxylase (TH) gene with CMV promoter was constructed and propagated. Southern blot analyses was used to identify positive plaques. Virus titer was about 1.4x10(14) pfu/L as determined by plaque forming assay. In glial cells infected with AdCMV th, the TH expression was demonstrated by immunohistochemical staining and HPLC-ECD. 678.8 ng DA was detected in the extract of 1x10(6) AdCMV th infected glial cells, but no detectable DA was found in AdCMVLacZ-infected glial cells. Injection of AdCMV th (1x10(7) pfu/rat) into the striatum of PD rats significantly improved the apomorphine-induced rotation movement(approximately 60%). The improvement in rotation movement remained up to 5 months after injection, and TH expression positive cells were found in the vicinity of injection. These results indicate that adenovirus may be a useful carrier for in vivo gene therapy in the PD patients.

Isolation and Characterzation of p160.2, a Signal Transducing Protein Downstream of Jak3.

Nonreceptor tyrosine kinase Jak3, which binds to intracellular domain of interleukin-2 receptor gamma subunit (IL-2Rgamma), plays an important role in IL-2 function. To find downstream signal transducer of Jak3, the N-terminal region JH3-JH7 of Jak3 was used as a baitg to screen a cDNA library by using the yeast two-hybrid system. One real true clone was obtained out of 10(5) cotransformants. Sequencing indicated that it encoded a p160.2 protein fragment which consisted of 399 amino acid residues. Further results showed that the p160.2 fragment merely bound to the intact JH3-JH7 through its C-terminal. Northern blot revealed that human p160.2 shared homology with monkey p160.2, but was different from mouse p160.2; and p160.2 was widely expressed in various tissues.

Cloning of Glial Cell Line-derived Neurotrophic Factor Gene and Its Expression in Eukaryotic Cells.

Glial cell line-derived neurotrophic factor gene (gdnf) was obtained by RT-PCR method. Its pro-duct in COS7 cells showed neurotrophic and protective effect on the dopaminergic neurons. Then gdnf gene was cloned into retrovirus vector pLNCX and packaged with PA317. Infectious particles thus obtained were used to infect rat myoblast cell line L-6TG. A cell clone L-6TG/gdnf was obtained after G418 selection. It will be applied to the ex vivo gene therapy study of Parkinson's disease.

Preliminary Study on Gene Therapy of PD Monkey Using Microcapsulated Rat Transgenetic Myoblasts.

Rat myoblast cells transfected with tyrosine hydroxylase gene were microcapsulated using the ALG/PLL system. These microcapsulated cells could survive grow and produce tyrosine hydroxylase(TH)for at least two months in vitro. After the implantation of these microcapsules into monkey striatum microcapsulated cells still survived one month more. No obvious gliosis was seen around implanted microcapsules. Then the microcapsules were transplanted into the striatum of PD monkeys and their typical PD rotation numbers were significantly decreased.

Expression of Brain-derived Neurotrophic Factor (BDNF) Gene in Rat Myoblast Cells.

Brain-derived neurotrophic factor (BDNF) gene was cloned into retrovirous vector plasmid pLNCX to form the pLNC/BDNF. After packaging in PA317 cell line, the infectious particles were used to infect rat myoblast cell line L-6TG. After selection with G418, the L-6TG/BDNF cells were collected. Results from Southern blot show that bdnf gene has successfully integrated into the L-6TG genomic DNA. The expression of bdnf gene has been proved by Northern blot and Dot blot. The content of BDNF in the culture medium of L-6TG/BDNF was about 25 ng.10(-6) cells per 24 h per ml.

Specific Expression of Suicide Gene in Liver Cancer Cells Mediated by Adenovirus.

Shuttle plasmid containing HSV-tk gene and regulatory sequence of the afp gene was constructed and recombined with the right arm of adenovirus DNA. The recombinant adenovirus vector was named pAdrAFPTK. Meanwhile, an AdCMVTK was constructed as control in which the tk gene was controlled under CMV promoter. PCR and Southern blot analyses were used to identify positive plaques. Virus titer was about 1x10(15) pfu/L determined by plaque forming assay. The AFP-positive cells or AFP-negative cells were infected with AdCMVTK or AdrAFPTK and then treated with GCV, respectively. Cytotoxic effects were assayed with MTT method. The IC(50) of GCV for both HeLa cells or BRL-3A cells (both were AFP-negative cells) and HepG2 cells (AFP-positive cells) were 1.3 &mgr;mol/L, 2&mgr;mol/L and <1&mgr;mol/L, respectively, after infection with AdCMVTK (m.o.i.=100). However, in the cases of infection with AdrAFPTK (m.o.i.=100), IC(50) were 1 000 &mgr;mol/L, >1 000 &mgr;mol/L and <1&mgr;mol/L for HeLa cells, BRL-3A cells and HepG2 cells, respectively. Results showed that this vector possessed advantages of high title, high infectivity coming from adenovirus and the character of cell type-specificity gene expression. The AdrAFPTK/GCV system may become a new, potent and specific approach for the gene therapy of the primary hepatoma.