Effects of pyraclostrobin on growth,oxidative stress,and gene expression in relation to stress and ATP-binding cassette transporters in Tetrahymena thermophila

Pyraclostrobin(PYR),a widely used fungicide,has negative effects on fish and algae,but its toxicity in protozoa remains unclear.In this study,the effects of PYR on the growth,oxidative stress,and gene expression related to stress and ATP-binding cassette(ABC)transporters in Tetrahymena thermophila were investigated.The result showed that the 96-h IC_(50)of PYR against T.thermophila was 17.2 mg/L.Moreover,PYR inhibited the growth of T.thermophila in concentration-or time-dependent manner.A morphological study revealed that the shape and size of T.thermophila changed,and damage of cell membrane surface was observed by scanning electron microscopy after 96 h of PYR exposure.The activities of superoxide dismutase(SOD)and catalase(CAT)increased throughout the experiment.In contrast,the glutathione(GSH)content was increased at 24 h and 48 h of exposure and decreased at 96 h.Moreover,a significant increase in malondialdehyde(MDA)level was observed in T.thermophila after96 h of exposure.Furthermore,PYR upregulated the HSP703,HSP705,GPx2,and ABAC15 gene expression in the 0.1–5-mg/L groups and downregulated the HSP704,HSP90,TGR,and ABCC52 mRNA levels at 96 h of exposure.These results suggest that PYR may exert adverse effects on T.thermophila by inducing oxidative stress and changing the gene expression related to ABC transporters and stress,which may enrich the understanding of the toxicity mechanism of PYR in aquatic organisms and provide reference data for aquatic ecological risk assessments.

Expression of the Human Growth Hormone Genein Insect Cells

The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control of the polyhedron gene promoter. The Spodoptera frugiperda (Sf9) cells was cotransfected with the plasmid DNA containing hGH gene and wildtype AcNPV DNA. The hGH gene was transferred to the AcNPV genome DNA through homologous recombination, and the recombinant virus rAcVhGH was obtained by multiple plaque purification. The high level of production of hGH (40 μg/mL) in supernatant of the infected monolayer culture was determined by immunochemiluminescent assay.

Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

AIM: To investigate the effect of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expression of epidermal growth factor receptor (EGFR) gene in gastric mucosal cells. METHODS: The stress-induced gastric mucosal injury in rat model was established by water-immersion and restrained stress methods. 52 rats were randomly divided into: normal group (n = 8), model group (n = 8), model serum group (n = 12), stomach serum group (n = 12), and gallbladder serum group (n = 12). The gastric mucosal cells were separated by pronase-EDTA digestion method and incubated with serum. The EGFR gene expression in gastric mucosal cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Compared with normal group (0.6860 ± 0.0594), the serum derived from rats of the stomach group (1.2272 ± 0.0813, P = 0.00 < 0.01) and gallbladder group (0.9640 ± 0.0387, P = 0.00 < 0.01) had a tendency to enhance the EGFR gene expression in gastric mucosal cells. Such tendency existed in the model group (0.7104 ± 0.0457) but with no signifi cant difference (P = 0.495 > 0.05) and in model serum group (0.8516 ± 0.0409) with an extremely obvious difference (P = 0.001 < 0.01). Furthermore, the EGFR gene expression in stomach serum group was significantly higher than that in gallbladder serum group (P = 0.00 < 0.01). CONCLUSION: The present study shows that serumderived from rats treated with electroacupuncture at stomach meridian acupoints can distinctly increase the EGFR gene expression of gastric mucosal cells. Therefore, there is certain meridian specificity in the serum, which could provide a proof for the TCM theory “particular relation between meridian and internal organ”.

Association between endogenous gene expression and growth regulation induced by TGF-β1 in human gastric cancer cells

AIM: To investigate the association between endogenous gene expression and growth regulation including proliferation and apoptosis induced by transforming growth factor-pi (TGF-βl) in human gastric cancer (GC) cells. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the main components of the TGF-β1/Smads signal pathway in human poorly differentiated GC cell line BGC-823. Localization of Smad proteins was also determined using immunofluorescence. Then, the BGC-823 cells were cultured in the presence or absence of TGF-β1 (10 ng/mL) for 24 and 48 h, and the effects of TGF-β1 on proliferation and apoptosis were measured by cell growth curve and flow cytometry (FCM) analysis. The ultrastructural features of BGC-823 cells with or without TGF-β1 treatment were observed under transmission electron microscope. The apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase (TdT)-mediated dTUP in situ nick end-labeling (TUNEL) method. Meanwhile, the expression levels of endogenous p15, p21 and Smad7 mRNA and the corresponding proteins in the cells were detected at 1, 2 and 3 h after culture in the presence or absence of TGF-β1 (10 ng/mL) by semi-quantitative RT-PCR and Western blot, respectively. RESULTS: The TGF-β1/Smad signaling was found to be intact and functional in BGC-823 cells. The growth curve revealed the most evident inhibition of cell proliferation by TGF-β1 at 48 h, and FCM assay showed G1 arrest accompanied with apoptosis induced by TGF-β1. The typical morphological changes of apoptosis were observed in cells exposed to TGF-β1. The apoptosis index (AI) in TGF-β1-treated cells was significantly higher than that in the untreated controls (10.7±1.3% vs 0.32±0.06%, P＜0.01). The levels of p15, p21 and Smad7mRNA and corresponding proteins in cells were significantly up-regulated at 1 h, but gradually returned to basal levels at 3 h following TGF-βl (10 ng/mL) treatment. CONCLUSION: TGF-β1 affects both proliferation and apoptosis of GC cells through the regulation of p15 and p21, and induces transient expression of Smad 7 as a negative feedback modulation of TGF-β1 signal. Our results suggest a novel functional role of p21 as an accelerant of TGF-β1-mediated apoptosis in GC cells.

EFFECT OF INTERLEUKIN-1β ON GROWTH HORMONE GENE EXPRESSION AND ITS POSSIBLE MOLECULAR MECHANISM IN RAT MtT/S SOMATOTROPH CELLS

Objective To elucidate the effect of interleukin-1β （IL- 1β） on human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S. Methods Stably transfected MtT/S cells were firstly established by transfecting 484-Lucl plasmid which contained hGH gene promoter --484 to ＋30 bp and luciferase reporter gene. The effect of IL-1β on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells. Results The 10^3 U/mL IL-1β stimulated secretion and synthesis of GH, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40 μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5 μmol/L） completely blocked the stimulatory effect of IL-1μ, and phosphatidylinositol-3-kinase （PI3-K） inhibitor LY294002 partly abolished the effect of IL-1μ. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit- 1 nor inhibition of Pit- 1 expression affected induction of hGH promoter activity by IL-1μ. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1β, and results showed that the stimulatory effect of IL-1β was abolished following deletion of the --196 to -- 132 bp fragment. Conclusions IL-1β promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1β on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.

MEK AND p38 MAPK-DEPENDANT PATHWAYS ARE INVOLOVED IN THE POSITIVE EFFECT OF INTERLEUKIN-6 ON HUMAN GROWTH HORMONE GENE EXPRESSION IN RAT MtT/S SOMATOTROPH CELLS

Objective To investigate the effect of interleukin-6(IL-6)on the human growth hormone(hGH)gene expression in a rat somatotropic pituitary cell line MtT/S.Methods The plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed.Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3.1(+)with DMRIE-C transfection reagent.After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways,the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.Results The 103 U/mL IL-6 stimulated GH secretion and synthesis,and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control.Among inhibitors of signaling transduction pathways,mitogen-activated protein kinase kinase(MAPKK/MEK)inhibitor PD98059(40 μmol/L)and p38 mitogen-activated protein kinase(MAPK)inhibitor SB203580(5 μmol/L)completely blocked the stimulatory effect of IL-6.Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells.Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity.A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6.The results showed that the stimulatory effect of IL-6 was abolished following deletion of the-196 to-132 bp fragment.Conclusions IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells.The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK,and a fragment of promoter sequence that spans the-196 to-132 bp of the gene,but may be unlinked with Pit-1 protein.

Effects of trichostatin A(TSA)on growth and gene expression in HeLa cells

Objective： To investigate the expressions of p53, RB1, Fas, c-fos, Ras, EGFR mRNA in human cervical cancer （HeLa） cell in response to the trichostatin A （TSA）. Methods： We took count of HeLa cells in different incubation times with TSA （0.2μm/L）. The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h. Then, we investigated the genes expression （mRNA levels） of HeLa cells after treatment for 36 h using SYBR green real-time PCR. Results： We demonstrated that trichostatin A （TSA） could make human cervical cancer （HeLa） cell morphological change and induce HeLa cell apoptosis. Furthermore, the data suggest that TSA-induced down-regulation of p53, RB1, Fas, but upregulated c-fos gene expression after treatment for 36 h, and Ras, EGFR did not show obvious response to TSA treatments. Conclusion： TSA has different effects on gene expression.

GENE EXPRESSION OF GROWTH FACTOR RECEPTORS AND NEURONAL MARKERS IN NEURO BLASTOMA CELL LINES

Gene expression of nerve growth factor receptor (NGFR), epidermal growth factor receptor (EGFR), chromogranin A (CGA) and neuropetide Y (NPY)in 4 aeuroblsstoma cell lines without N-myc amplification was studied by wins Northern blot technique, N type cells expressed more NGFR mRNA than S type cells and have only little or no EGFR expression. S type cells had stronger expression of EGFR mRNA than that of N type cells accompanying with only less or even no NGFR expression. The results Indicated that difference of gene expression of theae growth factor receptors might be due to the various of tumor cell differetiation. Celli differentiating toward neurons gave more NGFR expression and cells prepared to be differentiating toward other direction might give more EGFR gene expression.Various gene expression of CGA and NPY In neuroblsstoma cell lines might be due to the presence of different stages of tumor cell differentiation and NGF only Induced differentiation of those neuroblastoma cells ready to be differentiation to neurons afterwards.

Expression of Porcine Growth Hormone Gene in CHO Cell

An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Differential Expression of APOEGene in Porcine Fetal Fibroblast Cells

[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.

Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues

AIM To study the changes of gene expression of hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFr) in hepatocellular carcinoma (HCC) tissue and nontumorous liver tissue and the relationship between these changes and the biological behavior of the tumor.METHODS Gene expression of HGF and HGFr in 26 cases of HCC tissue and their adjacent nontumorous liver tissues was determined with digoxigenin-labeled DNA probes.RESULTS Positive expression of HGF in HCC tissue was similar to that in the adjacent nontumorous liver tissue, but positive rate of HGF expression was lower than HGFr gene expression. However, HGFr expression was higher in the metastatic cases than in those without metastasis. It was found that HGFr was overexpressed in HCC tissue as well as in the adjacent nontumorous liver tissue.CONCLUSION There seems to be a close relationship between overexpression of HGFr gene and tumor metastasis, and the HGF and HGFr system plays an important role in regulating tumor growth and metastasis.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation,cell death and nociception

Subsequent to a peripheral nerve injury, there are changes in gene expression within the dorsal root ganglia in response to the damage. This review selects factors which are well-known to be vital for inflammation, cell death and nociception, and highlights how alterations in their gene expression within the dorsal root ganglia can affect functional recovery. The majority of studies used polymerase chain reaction within animal models to analyse the dynamic changes following peripheral nerve injuries. This review aims to highlight the factors at the gene expression level that impede functional recovery and are hence are potential targets for therapeutic approaches. Where possible the experimental model, specific time-points and cellular location of expression levels are reported.

Differences in phenotype and gene expression of prostate stromal cells from patients of varying ages and their influence on tumour formation by prostate epithelial cells

Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Effects of Dietary Corn Gluten Meal on Growth Performance and Protein Metabolism in Relation to IGF-I and TOR Gene Expression of Juvenile Cobia (Rachycentron canadum)

A growth experiment was conducted on cobia(Rachycentron canadum,initial weight 108.2 g ± 3.0 g) to investigate the effects of dietary corn gluten meal(CGM) levels on the fish growth,whole body composition and protein metabolism in relation to specific gene expression.Five isonitrogenous(crude protein 45%) and isoenergetic(gross energy 20 kJ g 1) practical diets were formulated by replacing 0%(the control),17.5%,35.0%,52.5%,and 70.0% of fish meal(FM) protein with CGM protein.No significant differences were observed in the survival,feed intake(FI),specific growth rate(SGR),feed efficiency(FE) and protein productive value(PPV) among fish fed diets with 0%,17.5%,35.0%,and 52.5% of CGM protein.However,these indices were significantly lower in fish fed the diet with 70.0% of CGM protein than those in fish fed the control diet(P < 0.05).The whole-body crude protein and lipid contents were significantly lower while the whole-body moisture content was significantly higher in fish fed the diet with 70.0% of CGM protein compared with the control group(P < 0.05).When 70.0% of FM protein was replaced by CGM,plasma total protein and cholesterol contents were significantly lower than those in the control group(P < 0.05).Fish fed the diet with 70.0% of CGM protein had significantly lower hepatic insulin-like growth factor I(IGF-I) expression levels than those in the control group(P < 0.05).However,no significant differences were observed in hepatic target of rapamycin(TOR),dorsal muscle IGF-I and TOR expression levels among dietary treatments.Results of the present study indicated that 52.5% of FM protein could be replaced by CGM in the diets without significant influences on the growth,feed utilization and protein metabolism of juvenile cobia.The present results might be useful for developing cost effective and sustainable cobia dietary formulations.