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Biomedical subjects

Lin Wang

Publications and source records attributed to Lin Wang.

At least 181 records · Page 10Linked to original sources
PubMed2005

[Screening and cloning of hepatitis C virus non-structural protein 4A interacting protein gene in hepatocytes].

OBJECTIVE: To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A). METHODS: Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods. RESULTS: Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase. CONCLUSIONS: Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins↗
PubMed2005

[Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes].

OBJECTIVE: To investigate biological functions of non-structural protein 4B (NS4B) of hepatitis C virus (HCV), yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B. METHODS: HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT7 and transformed into yeast cells AH109 (type alpha). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics. RESULTS: Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit III, one retinol binding protein 4, one reticulon 3-A (RTN3) and one fibrinogen gamma polypeptide (FGG). CONCLUSION: Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.

Blotting, Western↗
PubMed2005

Effect of nitric oxide on toll-like receptor 2 and 4 gene expression in rats with acute lung injury complicated by acute hemorrhage necrotizing pancreatitis.

BACKGROUND: Toll-like receptor (TLR) 2/4 might play important roles in mediating proinflammatory cytokine synthesis and release. And nitric oxide (NO) has been used to treat acute respiratory distress syndrome (ARDS). This study aimed to investigate the changes in TLR2/4 gene expression in the lungs of rats with acute lung injury (ALI) complicated by acute hemorrhage necrotizing pancreatitis (AHNP) and the effect of NO on the TLR2/4 gene expression. METHODS: One hundred and ten SD male rats were randomly divided into sham-operated group (n=10), AHNP group (n=30), chloroquine-treated group (n=30), and L-Arg-treated group (n=40). The lungs were dissected for lung histological scoring, and bronchoalveolar lavages were harvested for lung injury indexing. TLR2/4 mRNA expression in the lungs was measured by RT-PCR. RESULTS: TLR2/4mRNA was detected in the lungs with low values in the sham-operated group (0.016+/-0.210E-2, 0.112+/-0.750E-2), but it was markedly increased at 3 hours in the AHNP group (0.787+/-0.751E-2, 1.512+/-1.794E-2), peaking at 12 hours (1.113+/-6.141E-2, 2.957+/-2.620E-2; P<0.05 or P<0.01). When lung injuries were aggravated, TNF-alpha concentrations in the lungs were increased, but NO concentrations were decreased (P<0.05 or P<0.01). When TLR2/4mRNA was inhibited by CQ (3h: 0.313+/-5.491E-2, 0.005+/-1.419E-3; 6h: 0.488+/-7.442E-2, 0.010+/-1.518E-3; 12h: 0.883+/-8.911E-2, 0.024+/-2.760E-3; P<0.05 or P<0.01), lung injuries were relieved. NO concentrations in the lungs were increased but TNF-alpha concentrations were decreased (P<0.05 or P<0.01). When the rats with AHNP were treated with L-Arg, TLR2/4mRNA expression in the lungs could be effectively inhibited (50 mg-T: 0.656+/-3.977E-2, 1.501+/-6.111E-2; 100 mg-T: 0.260+/-0.891E-2, 0.732+/-5.135E-2; 200 mg-T: 0.126+/-0.914E-2, 0.414+/-1.678E-2; 400 mg-T: 0.091+/-0.399E-2, 0.287+/-0.176E-2;P<0.05 or P<0.01) and lung injuries were relieved. At the same time, NO concentrations in the lungs were markedly increased, but TNF-alpha concentrations were decreased (P<0.05 or P<0.01). CONCLUSIONS: The expression of TLR2/4mRNA is increased in the lungs in rats with AHNP and lung injuries are aggravated.TLR2/4mRNA gene expression of the lungs of rats with AHNP could be markedly inhibited by NO, leading to the relief of lung injuries.

Animals↗
PubMed2005

[Effects of short-term VDT usage on visual functions].

OBJECTIVE: To evaluate the effects of short-term VDT usage on visual functions. METHODS: Visual function of the video display terminal (VDT) operators before and after 1 hour VDT usage were assessed. These included refraction, amplitude of accommodation, near point of convergence, AC/A, phorias, fusional convergence and divergence, tear film break time (BUT) and aberrations. And the same situation was compared was the non-VDT usage. RESULTS: In comparison with pre-VDT use, amplitude of accommodation, BUT were significantly decreased (P < 0.05), near point of convergence, near lateral exophoria were significantly increased after VDT use (P < 0.05). Total, high order, 4th and 5th order aberrations were significantly greater after VDT use (P < 0.05). CONCLUSIONS: Short-term VDT work does have a significantly greater temporarily effect on visual function, tear film quality and visual quality.

Adolescent↗
PubMed2005

[Development of WY21A interference fringe visuometer and its clinical applications].

WY21A Interference Fringe Visuometer, based on Thomas Young's interference theory, uses grating to produce interference lamp-house. This compact, flexible machine could be applied to measure interference fringe visual acuity. Its clinical tryout shows that WY21A is suited for daily practice as it is able to give a fast, easy and reliable preoperative assessments and prediction of visual recovery for most age-related cataract patients.

Equipment Design↗
PubMed2005

[Adsorptive properties of various carriers on dibutyl phthalate degradation bacteria].

OBJECTIVE: Seeking cheap and effective carriers for adsorbing dibutyl phthalate (DBP) degradation dominant bacteria. METHODS: Diatomite, clinoptilolite, silk zeolite and coal fly ash were selected as reserve materials and modified. The adsorptive capacity and intensity of each carrier to DBP degradation bacteria were measured. RESULTS: The adsorptive capacity of coal fly ash, clinoptilolite, silk zeolite and diatomite to DBP degradation dominant bacteria was 1.66, 1.37, 1.49 and 1.16 times in turn to that of natural carriers. The comparative tests for each modified carrier indicated that the adsorptive capacity of diatomite, clinoptilolite, silk zeolite and coal fly ash was 94.4%, 84.0%, 71.6% and 44.2% in turn. The adsorptive intensity of above carriers was 91.1%, 90.1%, 90.5% and 72.1%. The degradation rate to 100 mg/L DBP in 24h was 90.2%, 89.9%, 76.3% and 62.4% successively. CONCLUSION: The adsorptive capacity of diatomite, clinoptilolite, silk zeolite, and coal fly ash to DBP degradation dominant bacteria was enhanced after being modified. The adsorptive capacity and intensity from strong to weak was diatomite, clinoptilolite, silk zeolite, and coal fly ash.

Adsorption↗
PubMed2004

Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer.

AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, to detect human telomerase reverse transcriptase (hTERT) messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and clinicopathological parameters in patients with gastric cancer. METHODS: A real-time quantitative RT-PCR (RQ-PCR) based on TaqMan fluorescence methodology and the LightCycler system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100X (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, chi2 test and Fisher's exact test. RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27+/-0.89 and 0.93+/-0.18, respectively (t = 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation. CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

Adult↗
PubMed2004

Placement of 125I implants with the da Vinci robotic system after video-assisted thoracoscopic wedge resection: a feasibility study.

PURPOSE: To evaluate the feasibility of using the da Vinci robotic system for radioactive seed placement in the wedge resection margin of pigs' lungs. METHODS AND MATERIALS: Video-assisted thoracoscopic wedge resection was performed in the upper and lower lobes in pigs. Dummy (125)I seeds embedded in absorbable sutures were sewn into the resection margin with the aid of the da Vinci robotic system without complications. In the "loop technique," the seeds were placed in a cylindrical pattern; in the "longitudinal," they were above and lateral to the resection margin. Orthogonal radiographs were taken in the operating room. For dose calculation, Variseed 66.7 (Build 11312) software was used. RESULTS: With looping seed placement, in the coronal view, the dose at 1 cm from the source was 97.0 Gy; in the lateral view it was 107.3 Gy. For longitudinal seed placement, the numbers were 89.5 Gy and 70.0 Gy, respectively. CONCLUSION: Robotic technology allows direct placement of radioactive seeds into the resection margin by endoscopic surgery. It overcomes the technical difficulties of manipulating in the narrow chest cavity. With the advent of robotic technology, new options in the treatment of lung cancer, as well as other malignant tumors, will become available.

Animals↗
PubMed2004

Expression of the tumor suppressor gene ARHI in epithelial ovarian cancer is associated with increased expression of p21WAF1/CIP1 and prolonged progression-free survival.

PURPOSE: ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization. EXPERIMENTAL DESIGN: Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma. RESULTS: Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival. CONCLUSIONS: Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).

Adult↗
PubMed2004

Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation.

AIM: To investigate the effects of herbal compound 861 (Cpd 861) on cell proliferation in human hepatic stellate cells (LX-2) and human hepatocellular liver carcinoma cells (HepG2), and expression of alpha-smooth muscle actin (alpha-SMA) in LX-2 cells. METHODS: LX-2 and HepG2 cells were incubated with various concentrations of Cpd 861 (0.1-0.003 mg/mL) for 1, 2, 3, 5 and 7 d. Cell proliferation was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects of Cpd861 on the expression of alpha-SMA mRNA in LX-2 cells were measured by real-time quantitative PCR method using SYBR Green I technology. RESULTS: Cpd 861, at 0.1 mg/mL, significantly inhibited LX-2 cell proliferation (15% decrease relative to control, P<0.05) after 3 d of incubation. The inhibitory effects seemed to increase with the treatment time (25% decrease after 5 d of incubation and 35% decrease after 7 d of incubation, P<0.01). However, Cpd 861 did not affect HepG2 cell proliferation at the same concentration used for LX-2 cells. The expression levels of alpha-SMA mRNA decreased significantly when LX-2 cells were exposed to Cpd 861 for 48 h (59% decrease relative to control, P<0.05) or 72 h (60% decrease relative to control, P<0.01). CONCLUSION: Cpd 861 can significantly inhibit LX-2 cell proliferation in a dose-dependent manner, and reduce the expression levels of alpha-SMA mRNA in LX-2 cells. Since hepatic cell proliferation and high level of alpha-SMA are associated with liver fibrosis, the results suggest that Cpd 861 may be useful in the treatment of this disease.

Carcinoma, Hepatocellular↗
PubMed2004

Major histocompatibility class II transactivator (CIITA) mediates repression of collagen (COL1A2) transcription by interferon gamma (IFN-gamma).

Interferon gamma (IFN-gamma) plays an important role during inflammation by repressing collagen and activating major histocompatibility class II (MHC-II) expression. Activation of MHC-II by IFN-gamma requires regulatory factor for X-box 5 (RFX5) complex as well as class II transactivator (CIITA). We have shown that the RFX family binds to the COL1A2 transcription start site, and the RFX5 complex represses COL1A2 gene expression during IFN-gamma response. In this report, we demonstrate that CIITA is a key mediator of COL1A2 repression by IFN-gamma. IFN-gamma up-regulates the expression of CIITA in a time-dependent manner in lung fibroblasts and promotes CIITA protein occupancy on COL1A2 transcription start site in vivo as judged by chromatin immunoprecipitation (ChIP) assays. There are coordinate decreases in the occupancy of RNA polymerase II on the collagen transcription start site with increasing CIITA occupancy during IFN-gamma treatment. In addition, we are able to specifically knockdown the IFN-gamma-stimulated expression of CIITA utilizing short hairpin interference RNA (shRNA) against CIITA. This leads to the alleviation of COL1A2 repression and MHC-II activation by IFN-gamma. RFX5 recruits CIITA to the collagen site as evidenced by DNA affinity chromatography. The presence of RFX5 complex proteins enhances the collagen repression by CIITA reaching levels occurring during IFN-gamma treatment. Co-expression of CIITA with deletion mutations and collagen promoter constructs demonstrates that CIITA represses collagen promoter mainly through its N-terminal region including the acidic domain and the proline/serine/threonine domain. Our data suggest that CIITA is a crucial member of a repressor complex responsible for mediating COL1A2 transcription repression by IFN-gamma.

Animals↗
PubMed2004

Transactivating effect of hepatitis C virus core protein: a suppression subtractive hybridization study.

AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BamHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coli strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of beta-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by GenBank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.

Gene Expression Regulation, Viral↗
PubMed2004

Selection of potential markers for epithelial ovarian cancer with gene expression arrays and recursive descent partition analysis.

PURPOSE: Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers. EXPERIMENTAL DESIGN: To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed. RESULTS: The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF. CONCLUSIONS: Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.

Biomarkers, Tumor↗
PubMed2004

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells.

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.

Animals↗
PubMed2004

Stromal cells expressing elevated VCAM-1 enhance survival of B lineage tumor cells.

In the current study we generated murine bone marrow stromal cells that constitutively express human VCAM-1. These stromal cell lines allow investigation of the contribution of VCAM-1 initiated signaling to tumor cell survival. Co-culture of ALL cell lines with stromal cells engineered to over express VCAM-1 enhanced survival of leukemic cells in a PI-3 kinase-dependent manner, compared to co-culture with parental stromal cells expressing only endogenous VCAM-1. These observations suggest that modulation of stromal cell VCAM-1 by specific chemotherapeutic drugs may have utility in decreasing residual disease. In addition, these novel lines provide an in vitro model in which other tumor types that interact with stromal cells in the bone marrow microenvironment may be evaluated to determine the contribution of VCAM-1 initiated signaling to modulation of treatment response.

Animals↗
PubMed2004

Tunneling and coupled motion in the Escherichia coli dihydrofolate reductase catalysis.

H-transfer was studied in the complex kinetic cascade of dihydrofolate reductase. Intrinsic kinetic isotope effects, their temperature dependence, and other temperature-dependent parameters indicated H-tunneling, but no 1 degrees to 2 degrees coupled motion. The data also suggested environmentally coupled tunneling and commitment to catalysis on pre-steady-state isotope effects.

Catalysis↗
PubMed2004

Optimization of the Tet-on system to regulate interleukin 12 expression in the liver for the treatment of hepatic tumors.

Interleukin 12 (IL-12) is a potent antitumoral cytokine, but it can be toxic at high doses. Therapy of liver tumors might benefit from the use of vectors enabling tight control of IL-12 expression in hepatic tissue for long periods of time. To this aim, we have improved the Tet-on system by modifying the minimal region of the inducible promoter and adjusting the level of the trans-activator using liver-specific promoters with graded activities. The resulting vectors allowed hepato-specific gene regulation with lower basal activity and higher inducibility compared with the original system in the absence of repressor molecules. The basal and final protein levels depend on the strength of the promoter that directs the transcripcional activator as well as the relative orientation of the two genes in the same plasmid. We have selected the construct combining minimal leakage with higher level of induced gene expression to regulate IL-12 after DNA transfer to mouse liver. Administration of doxycycline (Dox) enhanced IL-12 expression in a dose-dependent manner, whereas it was undetectable in serum in the noninduced state. Gene activation could be repeated several times, and sustained levels of IL-12 were achieved by daily administration of Dox. The antitumor effect of IL-12 was evaluated in a mouse model of metastatic colon cancer to the liver. Complete eradication of liver metastasis and prolonged survival was observed in all mice receiving Dox for 10 days. These data demonstrate the potential of a naked DNA gene therapy strategy to achieve tight control of IL-12 within the liver for the treatment of cancer.

Animals↗