Semi-quantitative RT-PCR Assay Research Articles

Differential expression profile of growth hormone/chorionic somatomammotropin genes in placenta of small- and large-for-gestational-age newborns.

The human growth hormone/chorionic somatomammotropin (hGH/CSH) locus at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placenta-expressed genes (GH2, CSH1, CSH2, and CSHL1), is implicated in regulation of postnatal and intrauterine growth. A positive correlation has been reported between the offspring's birth weight and serum placental GH (coded by GH2) and placental lactogen (coded by CSH1, CSH2) levels in pregnant women. The objective of the study was the investigation of the hypothesis that the mRNA expression profile of placental hGH/CSH genes contributes to the determination of birth weight. We developed a sensitive, fluorescent-labeled semiquantitative RT-PCR assay coupled with gene-specific restriction analysis, capable of distinguishing alternative splice-products of individual placental hGH/CSH genes and quantification of their relative expression levels. The detailed profile of alternative transcripts of GH2, CSH1, CSH2, and CSHL1 genes in placenta from uncomplicated term pregnancies of the REPROMETA sample collection was addressed in association with the birth weight of newborns, grouped as appropriate for gestational age (AGA; n = 23), small for gestational age (SGA; n = 15), and large for gestational age (LGA; n = 34). The majority of pregnancies with SGA newborn showed down-regulation of the entire hGH/CSH cluster in placenta, whereas in the case of LGA, the expression of CSH1-1, CSH2-1, and CSHL1-4 mRNA transcripts in placenta was significantly increased compared with AGA newborns (P < 0.0001, P = 0.009, P = 0.002, respectively). The expression profile of placental hGH/CSH genes in placenta is altered in pregnancies accompanied by SGA and LGA compared with AGA newborns, and thus, it may directly affect the circulating fetal and maternal placental GH and placental lactogen levels.

Porcine lactoferrin administration enhances peripheral lymphocyte proliferation and assists infectious bursal disease vaccination in native chickens

The purpose of this study was to investigate the effects of dietary supplementation with recombinant porcine lactoferrin (rPLF) produced by yeast culture on peripheral lymphocyte proliferation and serum antibody titers in chickens vaccinated against the infectious bursal disease (IBD) virus. Treatment groups were fed with rPLF powder in their diet (2.0%, w/w), and the IBD vaccine was administrated at 1 and 3 weeks of age. At 8, 12, and 16 weeks after vaccination, serum IBD antibody titers were measured via the micro-method and T cell proliferation rates were evaluated. In gene expression analyses, rPLF-treated chicken peripheral T lymphocytes were stimulated with concanavalin A (ConA) for 24 h. The mRNA expression levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-12 (IL-12) were determined using a semi-quantitative RT-PCR assay. The results revealed that the rPLF additive led to significant increases in serum IgG and IBD-specific antibody titers ( P < 0.05). The rPLF administration significantly increased chicken intestinal villous lengths and also enhanced the expression of IFN-γ and IL-12 in chicken T lymphocytes. These data suggest that rPLF enhances cell-mediated immunity and augment the ability of IBD vaccination to benefit chicken industry in disease resistance.

Molecular cloning and immune responsive expression of MDA5 gene, a pivotal member of the RLR gene family from grass carp Ctenopharyngodon idella

The cytoplasmic helicase protein MDA5 (melanoma-differentiation-associated gene-5) recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 5′ triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp Ctenopharyngodon idella. The full length of the C. idella MDA5 ( CiMDA5) cDNA is 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (conserved restriction domain of bacterial type III restriction enzyme), two HELICc (helicase superfamily c-terminal domain) and an RD (regulatory domain). The CiMDA5 mRNA was widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by semi-quantitative RT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P < 0.05), reached the crest at 24 h (7.48 folds, P < 0.05), and then recovered to normal level at 48 h post-injection ( P > 0.05) of grass carp reovirus (GCRV). In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P < 0.05) and returned to control level at 72 h ( P > 0.05) after GCRV challenge. These data implied that CiMDA5 involved in the early stage of antiviral innate immune defense to GCRV in grass carp, and provided new insight into the evolution research of RLR (RIG-I like receptor) gene family.

Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples.

SURVEY OF THE EFFECT OF IRRIGATION PERIODS ON PDS AND BCH GENES EXPRESSION WITH RT-PCR IN SAFFRON

ISHS III International Symposium on Saffron: Forthcoming Challenges in Cultivation, Research and Economics SURVEY OF THE EFFECT OF IRRIGATION PERIODS ON PDS AND BCH GENES EXPRESSION WITH RT-PCR IN SAFFRON

The Effect of Atorvastatin and Simvastatin on NIS Expression of the TPC-1 Cell under the Therapeutic Blood Concentrations

Background: Although so many experimental trials have been done to improve the redifferentiation and responsiveness of radioiodide therapy, they have not yet yielded any satisfactory results. As statins inhibit both farnesylation and geranylgeranylation, they have been reported to have an antineoplastic and redifferentiation effect in experimental and clinical studies. In this study, we investigated the relationship between statins and the alteration of the NIS expression and, TPC-1 cell apotosis to evaluate the possibility of using statins as adjuvant therapeutic agents for papillary thyroid cancer. Methods: We used the TPC-1 cell lines for our experiments. Cell viabilities were measured by CCK-8. The degrees of apoptosis and, the expressions of NIS mRNA and NIS protein were measured by flow cytometry, semi quantitative RT-PCR and Western blot assay. Results: Increased levels of NIS mRNA and NIS protein were observed under therapeutic blood concentrations (concentrations of simvastatin: 20, 50, 80 nM, concentrations of atorvastatin: 50, 80,110 nM), but the dose-response relationship was only manifested within simvastatin. The TPC-1 cells showed a concentration dependent decrease of viability and an increase of apoptosis not under therapeutic blood concentrations, but under excessively high concentrations (after treatment with 10-50 of atorvastatin and with 1-10 of simvastatin). Conclusion: The results of this study show that effective therapeutic blood concentrations of simvastatin and atorvastatin can give a favorable effect on the NIS expression under effective therapeutic blood concentrations. Therefore, we demonstrated the possibility that simvastatin and atorvastatin might have an important role as adjuvant therapeutic agents to improve the responsiveness of radioiodide therapy for papillary thyroid cancer. Further studies are needed to clarify this issue.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

BRCA1 185delAG mutant protein, BRAt, up-regulates maspin in ovarian epithelial cells

Objective Aggressive clinical course and difficult detection of ovarian cancer are major challenges to improving patient survival and necessitate avid investigation into more effective therapeutic approaches. Understanding early molecular and pathological changes in high risk patients, such as BRCA1 mutation carriers, can provide candidates for molecular profiling and novel targets for effective therapies. Methods Using a culture model system for normal human ovarian surface epithelial cells with and without the BRCA1 185delAG frameshift mutation for the truncated protein product, BRAt, we investigated the role of BRAt in enhanced chemosensitivity. We used MTS, Western immunoblot, semi-quantitative RT-PCR, luciferase reporter and siRNA assays, to identify novel downstream targets of BRAt that promote apoptosis following chemotherapeutic treatment. Results We identified maspin as a novel downstream target of BRAt. BRAt increases maspin expression with preferential nuclear localization of maspin. Further, Brat-mediated maspin expression is transcriptionally regulated through an AP1 site within the (−520) to (−297) region of the promoter. Lastly, BRAt, enhances chemosensitivity in normal ovarian surface epithelial cells through c-Jun by a mechanism that may involve maspin. Conclusions BRAt-mediated enhanced chemosensitivity correlates clinically with enhanced chemotherapeutic response in BRCA1 mutation carriers. BRAt-mediated maspin expression also correlates with improved prognostic outlook for ovarian tumors with high levels of nuclear maspin. Consequently, understanding early genotypic and phenotypic changes in the context of high risk disease may provide a better understanding of the mechanism of mutation-associated ovarian cancer and provide new targets for therapeutic intervention.

Expression of a Glutathione Reductase from Brassica rapa subsp. pekinensis Enhanced Cellular Redox Homeostasis by Modulating Antioxidant Proteins in Escherichia coli

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H(2)O(2), menadione, and heavy metal (CdCl(2), ZnCl(2) and AlCl(2))-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H(2)O(2) stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.

The intravenous anesthetics barbiturates inhibit hypoxia-inducible factor 1 activation

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of barbiturates on HIF-1 activity has not been reported. In this study, we investigated the effect of thiopental and thiamylal on HIF-1 activity using the neuronal SH-SY5Y cells, the non-neuronal HEK293 cells, and the macrophage-differentiated THP-1 cells. Cells were exposed to 20% or 1% O 2 conditions with or without thiopental or thiamylal treatment. The cell lysate were subjected to Western blot analysis using anti-HIF-1α and -HIF-1β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time RT-PCR and luciferase assay. Hydroxylation of HIF-1α protein was evaluated by in vitro pulldown assay using recombinant protein. Both thiopental and thiamylal reversibly suppressed hypoxia-induced HIF-1 activation in the neuronal and the non-neuronal cells in a dose-dependent manner. Moreover, the barbiturates inhibited lipopolysaccharide-induced HIF-1α expression in THP-1 cells. The HIF-1-downstream gene expression was also inhibited by the barbiturates. HIFα-hydroxylases activity and HIF-1α stability were not affected but the HIF-1α protein neosynthesis was inhibited by the barbiturates. Our experimental results indicate that barbiturates inhibit induced HIF-1 activation and downstream genes expression.

Molecular Characterization of Rockbream (Oplegnathus fasciatus) Cytoskeletal β-actin Gene and Its 5'-Upstream Regulatory Region

The cytoskeletal <TEX>$\beta$</TEX>-actin gene and its 5'-upstream region were isolated and characterized in the rockbream (Oplegnathus fasciatus). Complementary DNA of the rockbream <TEX>$\beta$</TEX>-actin represented a 1,125 bp of an open reading frame encoding 375 amino acids, and the rockbream <TEX>$\beta$</TEX>-actin cDNA and deduced amino acid sequences were highly homologous to those of other vertebrate orthologs. At the genomic level, the <TEX>$\beta$</TEX>-actin gene also exhibited an organization typical of vertebrate cytoskeletal actin genes (2,159 bp composed of five translated exons interrupted by four introns) with a conserved GT/AG exon-intron splicing rule. The putative non-translated exon predicted in the rockbream <TEX>$\beta$</TEX>-actin gene was much more homologous with those of teleostean <TEX>$\beta$</TEX>-actin genes than those of mammals. The 5'-upstream regulatory region isolated by genome walking displayed conserved and essential elements such as TATA, CArG and CAAT boxes in its proximal part, while several other immune- or stress-related motifs such as those for NF-kappa B, USF, HNF, AP-1 and C/EBP were in the distal part. Semi-quantitative RT-PCR assay results demonstrated that the rockbream <TEX>$\beta$</TEX>-actin transcripts were ubiquitously but different-tially expressed across the tissues of juveniles.

Altered expression of the SEMA3B gene in epithelial tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

Effective inhibition of infectious bursal disease virus replication by recombinant avian adeno-associated virus-delivered microRNAs

RNA interference (RNAi) is a novel antiviral strategy against a variety of virus infections. Infectious bursal disease virus (IBDV) causes an economically important disease in young chickens. This study demonstrated efficient inhibition of IBDV replication by recombinant avian adeno-associated virus (rAAAV)-delivered anti-VP1 and anti-VP2 microRNAs (miRNAs). In the viral vector-transduced cells, sequence-specific miRNA expression was detected by poly(A)-tailed RT-PCR. Reporter assays using a pVP2-EGFP vector showed significant and long-lasting inhibition of VP2-EGFP expression in cells transduced with anti-VP2 miRNA-expressing rAAAV-RFPmiVP2E, but not with the control miRNA-expressing rAAAV-RFPmiVP2con or anti-VP1 miRNA-expressing rAAAV-RFPmiVP1. Semi-quantitative RT-PCR and/or virus titration assays showed a significant inhibitory effect on homologous IBDV replication in cells transduced with rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E. For two heterologous IBDV isolates, transduction with rAAAV-RFPmiVP1 led to slightly weaker but similar inhibitory effects, whereas transduction with rAAAV-RFPmiVP2E resulted in significantly weaker and different inhibitory effects. These results suggest that rAAAV could act as an efficient vector for miRNA delivery into avian cells and that VP1 is the more suitable target for interfering with IBDV replication using RNAi technology.

Overexpression of eukaryotic initiation factor 4E ( eIF4E) and its clinical significance in lung adenocarcinoma

Background Eukaryotic initiation factor 4E ( eIF4E), an important regulator of translation, plays important roles in tumor transformation, progression and metastasis. However, the clinical significance of eIF4E expression in lung adenocarcinoma (AdC) remains unclear. The aim of this study was to explore the expression of eIF4E gene in lung adenocarcinoma cell lines and tissues, and to investigate its relationship with clinical characteristics and prognosis of patients with lung adenocarcinoma in combination with p53 status. Methods Semi-quantitative RT-PCR and Western blotting assays were performed to detect the expression of eIF4E mRNA and protein in normal human lung epithelial cell line, immortalized lung epithelial cell line and lung adenocarcinoma cell lines. Additionally, the expression of eIF4E gene was also detected in 32 cases of lung adenocarcinoma tissues, tumor adjacent tissues and tumor surrounding normal tissues by the same methods. Moreover, expression of eIF4E and the status of p53 in specimens from 76 patients with lung adenocarcinoma were examined by immunohistochemical staining. Correlations between eIF4E expression and clinicopathological features, and the effect of eIF4E on prognosis of patients with lung adenocarcinoma were evaluated by statistical analysis. Results The levels of eIF4E mRNA and protein expression were higher in lung adenocarcinoma cell lines and in telomerase-immortalized lung epithelial cell line than in the normal lung epithelial cell line. The expression of eIF4E gene showed statistical difference between tumor tissues, tumor adjacent tissues and tumor surrounding normal tissues ( P < 0.05). Moreover, the higher levels of eIF4E expression were correlated with poorer differentiation ( P = 0.012), higher pathological stage ( P < 0.0001) and clinical stage ( P = 0.002), a higher incidence of hematogenous metastasis ( P = 0.007) and cancer-related death ( P = 0.036). The 5-year survival rate of patients with higher eIF4E expression was significantly lower than that of patients with lower eIF4E expression ( P = 0.0045). Furthermore, in a multivariate analysis by Cox regression model, high eIF4E expression was confirmed to be an independent prognostic factor (HR: 2.258; unfavorable, P = 0.0056), while lymph node (HR: 2.033; unfavorable, P = 0.0440) and hematogenous metastasis (HR: 3.489; unfavorable, P < 0.0001) were also significant prognostic factors. Conclusion High eIF4E expression was correlated with poorer overall survival in lung adenocarcinoma patients. eIF4E might be a better clinical marker predicting the prognosis for lung adenocarcinoma patients in combination with p53 status.

Expression of STK15 mRNA in hepatocellular carcinoma and its prognostic significance

Objectives To investigate whether the STK15 mRNA expression correlates with clinicopathologic features and the prognosis of HCC patients. Design and methods Three hepatoma cell lines, two normal liver epithelial cell lines, hepatoma tissues, adjacent tumor tissues and normal liver tissues were obtained from 46 HCC patients. Semi-quantitative RT-PCR assays were performed to detect the expression of STK15 mRNA in above cell lines and tissues. Moreover, the expression of STK15 protein in hepatoma tissues, adjacent tumor tissues and normal liver tissues was also examined by immunohistochemical staining. Finally, correlations between STK15 mRNA expression and the clinicopathological features and prognosis of HCC patients were evaluated. Results STK15 mRNA showed higher levels in hepatoma cell lines than in normal liver epithelial cell lines. Moreover, the mean levels of STK15 mRNA and protein expression showed statistical difference between tumor tissues, tumor adjacent tissues and normal liver tissues ( P < 0.01). By immunohistochemical analysis, we found that paraffin-embedded archival HCC tissues showed higher expression of STK15 than adjacent tumors and normal liver tissues. Furthermore, HCC patients with higher STK15 mRNA expression showed poorer prognosis than those with lower STK15 mRNA expression. The high level of STK15 mRNA expression was significantly correlated with tumor stage ( P = 0.0081), more frequent lymph node ( P = 0.0380) or hematogenous metastasis ( P = 0.0066), and a higher incidence of cancer-related death ( P = 0.0083). Furthermore, the disease-free survival (DFS) and overall survival (OS) rates of HCC patients with higher STK15 mRNA expression group (47.6% and 52.7%) were significantly lower than those of patients with low STK15 mRNA expression group (56.9% and 68.8%, P = 0.0018 and 0.0047). Conclusions STK15 mRNA might be a good marker for predicting the prognosis of HCC patients.

Vasoactive intestinal peptide behaves as a pro‐metastatic factor in human prostate cancer cells

There is little known on the involvement of vasoactive intestinal peptide (VIP) in the metastatic cascade of human prostate cancer, that is, cell proliferation, cell-cell adhesion, extracellular-matrix degradation, and migration/invasion. Here we evaluated the expression of related biomarker proteins (cyclin D1, metalloproteinases MMP-2 and MMP-9, and E-cadherin) in human androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells. Reverse transcriptase (RT)-polymerase chain reaction (PCR), gelatin zymography, Western blotting, confocal immunofluorescence microscopy, and assays on cell proliferation, adhesion, wound-healing, migration and random homing were performed. VIP increased cell proliferation and cyclin D1 expression whereas it decreased cell adhesion and E-cadherin expression in LNCaP and PC3 cells. VIP enhanced the gelatinolytic activity of MMP-2 and MMP-9. Semiquantitative RT-PCR assays showed that VIP stimulated mRNA levels of these MMPs and suppressed mRNA levels of its inhibitory protein RECK. VIP promoted cell invasion and migration, and the responses were faster according to the most aggressive status in cancer progression (androgen-independence). The involvement of nuclear factor-kappaB (NF-kappaB) was demonstrated since the anti-inflammatory agent curcumin blocked VIP effects on the above biomarkers in both cell lines. Taken together, these results and the presence of kappaB sites on gene promoter of cyclin D1, MMPs and, possibly, E-cadherin suggest that VIP may act as a cytokine in an early metastatic stage of human prostate cancer through the NF-kappaB/MMPs-RECK/E-cadherin system. Our findings may help to define novel targets and agents with potential usefulness in prostate cancer therapy.

Induction of interleukin-8 production by angiotensin II in rat vascular smooth muscle cells

Objective Interleukin-8(IL-8) represents the prototypical chemokine that is made by a wide variety of cell types. Previously studies have suggested that angiotensin II(Ang II) is involved in atherogenesis through induction of proinflammatory cytokines such as interleukin-6 or monocyte chemoattractant protein-1(MCP-1) in vascular smooth muscle cells(VSMCs), while the role of Ang II on IL-8 expression in VSMCs is poorly studied. Methods In this study, VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats. The expression of smooth muscle α-actin was confirmed by an immunohistochemical method. Semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) analyses were conducted to detect IL-8 expression. Results In the present study we found that Ang II significantly increased the expression of IL-8 both at the mRNA and protein levels in rat VSMCs in a dose- and time-dependent manner. Conclusion These findings suggested that Ang II may participate in atherosclerosis through induction of inflammatory mediator in VSMCs.

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7

Aims The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. Main methods MCF-7 was treated with EGCG (20 μM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. Key findings EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors α5, β1, αv and β3 due to EGCG treatment. Significance Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Expression of cathepsin L gene of fasciola gigantica in different developmental stages

5 Abstract: Fascioliasis is one of the most serious parasitic infestations of livestock. It is caused by Fasciola he patica (Temperate re gions) and Fasciola gi gantica (Tropical re gions).It also has a zoonotic importance. The proteolytic enzyme; ca thepsin-L cysteine proteinase (28-28.5 KDa) h elps the parasite in tissue i nvasion, host immune evasion and nutrients intake. This study aimed to check weather Fasciola gigantica flukes d epend upon cathepsin-L throughout t heir life c ycle in snail and mammalian hosts or n ot. For th at ca thepsin-L gene was inv estigated in adult and other d evelopmental stages o f Fasciola gi gantica using semi-quantitative RT-PCR assay. C athepsin-L protein w as d etected by us ing Sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) anal ysis and detection of cathepsin-L cys teine proteinase antigen in examined stages by Enzyme linked immunotransfer blot (EITB) analysis. Stages of Fasciola gi gantica were collected from an experimental l ife cycle that wa s c ompleted in our la boratory. The adult worms a nd eg gs were c ollected f rom bile d ucts and gal l bladders, respectively, of slaughtered naturally infected cattle at El-Shohada abattoir, Minufiya governorate, Egypt. The results obtained revealed that ca thepsin-L gene is expressed in adult and other d evelopmental stag es o f F asciola gigantica wi th a molecular weight of 28-28.5 kDa. These results may be promising for developing an effective and reliable method for e arly diagnosis of Fasciola gi gantica and protection cysteine proteinase enzyme of Fasciola gigantica is present in ad ult and other developmental stages with m olecular weight at 28-28.5 kDa and its gene is fully expressed in adult and other d evelopmental stag es.

The effect of p53 on the DNA repair enzyme Thymine-DNA Glycosylase

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.