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Transcription-polymerase Chain Reaction (transcription-polymerase + chain_reaction)
Kinds of Transcription-polymerase Chain Reaction Terms modified by Transcription-polymerase Chain Reaction Selected AbstractsDystrophin upregulation in pressure-overloaded cardiac hypertrophy in ratsCYTOSKELETON, Issue 1 2003Masato Maeda Abstract Dystrophin is a cytoskeletal protein localized to the sarcolemma of skeletal and cardiac muscle, and neurons. We have recently demonstrated that a significant cardiac damage including myocytes injury, inflammation, and fibrosis, was found in dystrophin-deficient myocardium during pressure overload [Kamogawa et al., 2001: Cardiovasc Res 50:509,515]. However, little is known about how the cardiac sarcolemmal cytoskeleton produces qualitative and quantitative changes in response to pressure overload. Accordingly, we investigated dystrophin gene expression and protein accumulation during cardiac hypertrophy. Cardiac hypertrophy was produced by banding of the abdominal aorta of rats. Total RNA from the left ventricle of the heart was used for a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Dystrophin mRNA expression significantly increased by 33 ± 18% at 1 day (P < 0.05) and 45 ± 19% at 2 days (P < 0.01) after banding, while G3PDH mRNA showed no significant change. RT-PCR for dystrophin tissue-specific exon 1 revealed that only muscle type promoter, but not non-muscle type promoter (brain and Purkinje-cell type), was activated immediately after banding. Immunohistochemistry for dystrophin showed intense cellular membrane staining with an increase in the perimeter of the myocytes by 14% at 3 days (46.3 ,m, P < 0.01) and 19% at 7 days (51.2 ,m, P < 0.01) after banding. Western blotting also showed dystrophin protein increased by 14 ± 6% at 2 days (P < 0.05) and by 32 ± 10% at 3 days (P < 0.01) after aortic banding. In conclusion, upregulation of dystrophin mRNA expression and protein accumulation occurs in response to cardiac hypertrophy. These data and the vulnerability of dystrophin-deficient myocardium to pressure overload suggest that dystrophin could play an important role in maintaining the integrity of the sarcolemma. Cell Motil. Cytoskeleton 55:26,35, 2003. © 2003 Wiley-Liss, Inc. [source] Effects of metformin and oleic acid on adipocyte expression of resistinDIABETES OBESITY & METABOLISM, Issue 1 2006R Rea Aim:, The adipocyte-secreted hormone resistin has been implicated in obesity-induced insulin resistance and type 2 diabetes, but pharmacological and dietary factors that regulate resistin gene expression and the effects of resistin on cellular glucose uptake in muscle have not been clearly defined. Methods:, Expression of resistin mRNA was studied in differentiated 3T3-L1 adipocytes by using real-time semiquantitative reverse transcription-polymerase chain reaction. The effects of resistin on insulin-stimulated and insulin-independent 2-deoxyglucose uptake were evaluated in L6 muscle cells. Results:, Insulin 1 µm and rosiglitazone 10 µm markedly reduced resistin mRNA expression (relative to the control gene TF2D) by 4.7-fold (p < 0.05) and 5.3-fold (p < 0.02), respectively. Similar reductions in resistin mRNA were demonstrated with metformin 100 µm (6.2-fold reduction, p < 0.02) and oleic acid 100 µm (3.9-fold reduction, p < 0.03). Resistin 1 µm significantly reduced maximum insulin-stimulated 2-deoxyglucose uptake in L6 cells from 634 to 383% (relative to 100% for control, p < 0.001), and co-administration of rosiglitazone had no effect on resistin-induced insulin resistance. In the absence of insulin, however, resistin increased glucose uptake dose-dependently (e.g., 1.75-fold at 5 µm, p < 0.001) via a mitogen-activated protein kinase-dependent pathway. Conclusions:, These results demonstrate that various glucose-lowering therapies and oleic acid reduce resistin gene expression in isolated adipocytes, and that resistin impairs insulin-stimulated glucose uptake in skeletal muscle-derived cells. [source] Expression of caspase and apoptotic signal pathway induced by sulfur dioxideENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2010Juli Bai Abstract Sulfur dioxide (SO2) is a common air pollutant that is released in low concentrations into the atmosphere and in higher concentrations in some work places. In the present study, male Wistar rats were housed in exposure chambers and treated with 14.00 ± 1.01, 28.00 ± 1.77, and 56.00 ± 3.44 mg/m3 SO2 for 7 days (6 hr/day), while control rats were exposed to filtered air under the same conditions. The mRNA and protein levels of caspase-3, caspase-8, and caspase-9 were analyzed using a real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay and an immunohistochemistry method. Activities of caspases were detected using colorimetric and fluorescent assays. Chromatin degradation and cell morphological changes were investigated by TUNEL assay and H&E staining in livers and lungs, respectively. The results showed that mRNA levels, protein levels and activities of caspase-3, caspase-8, and caspase-9 were increased in a dose-dependent manner in livers and lungs of rats after SO2 inhalation. In addition, livers were infiltrated with lymphocytes, congestion and inflammation occurred in lungs, and eosinophil cells and apoptotic cells increased in both livers and lungs after SO2 inhalation. These results suggest that SO2 exposure increases the expression and activity of both initiator and and effector caspases, and may induce apoptosis in liver and lung of rats through both death receptor and mitochondrial pathways. Environ. Mol. Mutagen. 2010. © 2009 Wiley-Liss, Inc. [source] Impact of microcystin containing diets on physiological performance of Nile tilapia (Oreochromis niloticus) concerning stress and growth,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Andrea Ziková Abstract Diets containing Microcystis with considerable amounts of the cyanotoxin microcystin-LR (MC-LR) were fed to determine their impact on the physiological performance of the omnivorous Nile tilapia (Oreochromis niloticus) with regard to stress and growth performance. Four different diets were prepared based on a commercial diet (control, MC-5% [containing 5% dried Microcystis biomass], MC-20% [containing 20% dried Microcystis biomass], and Arthrospira-20% [containing 20% dried Arthrospira sp. biomass without toxin]) and fed to female Nile tilapia. Blood and tissue samples were taken after 1, 7, and 28 d, and MC-LR was quantified in gills, muscle, and liver by using high-performance liquid chromatography (HPLC). Only in the liver were moderate concentrations of MC-LR detected. The stress hormone cortisol and glucose were analyzed from plasma, suggesting that all modified diets caused only minor to moderate stress, which was confirmed by analyses of hepatic glycogen. In addition, the effects of the different diets on growth performance were investigated by determining gene expression of hypophyseal growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I). For all diets, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) demonstrated no significant effect on gene expression of the major endocrine hormones of the growth axis, whereas classical growth data, including growth and feed conversion ratio, displayed slight inhibitory effects of all modified diets independent of their MC-LR content. However, no significant change was found in condition or hepatosomatic index among the various diets, so it seems feasible that dried cyanobacterial biomass might be even used as a component in fish diet for Nile tilapia, which requires further research in more detail. Environ. Toxicol. Chem. 2010;29:561,568. © 2009 SETAC [source] A comparison of molecular methods for the routine detection of viroids,EPPO BULLETIN, Issue 3-4 2000R. A. Mumford Viroids, such as Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd), are important plant pathogens. However, because of their unique biological properties, viroids have proved, in the past, difficult to diagnose. The use of molecular methods has now changed this and this paper reports the comparison of three such methods (dot-blot hybridization using DIG-labelled cRNA probes, reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan), which have been developed for routine detection of CSVd. Sensitivity comparisons show that the TaqMan assay is more sensitive than either RT-PCR (100 times) and hybridization (1000 times). RT-PCR and TaqMan assays have also been developed to detect PSTVd. In addition to the development of sensitive detection methods, considerable emphasis has been placed on making these assays amenable to mass-scale detection through the use of internal controls and the development of a rapid, reliable probe capture extraction system. [source] Evaluation of PG-M3 antibody in the diagnosis of acute promyelocytic leukaemiaEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2010Sanjeev Kumar Gupta Eur J Clin Invest 2010; 40 (10): 960,962 Abstract Background & objectives, Acute promyelocytic leukaemia (APL) is a distinct subtype of acute myeloid leukaemia (AML) characterized by a reciprocal translocation, t(15;17) and a high incidence of life-threatening coagulopathy. APL diagnosis is considered a medical emergency. As reverse transcription-polymerase chain reaction (RT-PCR) for PML-RAR, fusion oncoprotein is time consuming, there is a need for a rapid and accurate diagnostic test for APL. This study evaluates the role of PG-M3 monoclonal antibody using immunofluorescence (IF) in the early diagnosis of APL. Materials and Methods, Thirty-six new untreated APL cases diagnosed with RT-PCR for PML-RAR, as the gold standard and 38 non-APL controls (28 non-APL AMLs and 10 non-leukaemic samples) were evaluated by routine morphology and cytochemistry, RT-PCR and IF using PG-M3 monoclonal antibody. Results, Using IF, 34 of 36 (94·4%) APL cases showed a microgranular pattern suggestive of APL and two cases (5·6%) showed a speckled pattern typical of wild-type PML protein (False negative). By comparison, two of 28 (7·1%) non-APL AMLs showed microgranular pattern (false positive). Hence, IF as a diagnostic test for APL resulted in a sensitivity of 94·4%, specificity of 92·9% and positive and negative predictive values of 94·4% and 92·9% respectively. All 10 non-leukaemic samples showed a speckled pattern. Conclusions, IF using PG-M3 antibodies can be used as a rapid (takes 2 h), cheap, sensitive and specific method to identify APL. It can be a useful adjunct for diagnosis of APL especially if facilities for RT-PCR are not available, particularly in resource-limited settings. [source] Placenta growth factor stimulates the growth of Philadelphia chromosome positive acute lymphoblastic leukemia cells by both autocrine and paracrine pathwaysEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2005Toshiko Ikai Abstract:, Vascular endothelial growth factor (VEGF) and its associated molecule, placenta growth factor (PlGF) are now known to support normal hematopoiesis, and leukemia cell growth. In this study, expression of VEGF and PlGF in acute lymphoblastic leukemia (ALL) cells was examined by real time reverse transcription-polymerase chain reaction in 20 patient samples. Expression of PlGF was more intense in Philadelphia chromosome positive (Ph+) ALL than in Ph, ALL cases. On the other hand, expression level of VEGF was not different between Ph+ and Ph, cases. Then, PlGF was added to the two ALL cell lines, CRL1929 (Ph+), and Nalm6 (Ph,). The PlGF stimulated the growth of CRL1929 in time- and dose-dependent manners, although the growth of Nalm6 was not affected by PlGF. The growth stimulation of CRL1929 by PlGF was confirmed by the increase of S phase cells. And the growth promoting effect of PlGF on CRL1929 was cancelled by simultaneous addition of VEGFR1/Fc (which binds to PlGF and abrogates its function), but was not cancelled by VEGFR2/Fc (which does not bind to PlGF). Then, addition of VEGFR1/Fc to the simple culture of CRL1929 demonstrated growth inhibitory effect. These observations demonstrated that PlGF stimulates the growth of Ph+ ALL cells by both autocrine and paracrine pathways. Finally, PlGF-VEGFR1 loop might be a therapeutic target to improve the prognosis of Ph+ ALL. [source] Expression of DNA repair gene Ku80 in lymphoid neoplasmEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2005Tsai-Yun Chen Abstract:,Objectives:,Ku, a heterodimer of KU70 and Ku80 that binds to double-strand DNA breaks (DSBs) and activates the catalytic subunit (DNA-PKcs) when DNA is bound, is essential in DSB repair and V(D)J recombination. Ku80 is a putative tumor suppressor gene that might play an important role in drug resistance. Our aim was to determine the role of Ku80 in lymphoid malignancy. Patients and methods:,Competitive reverse transcription-polymerase chain reaction assays were performed and the expression levels of Ku80 were measured in normal peripheral blood mononuclear cells (n = 9) and malignant cells from 25 patients with acute lymphoblastic leukemia (ALL) (14 children, 11 adults), and chronic lymphoproliferative disorders (n = 6). The Ku80 transcripts were sequencing for the possibility of mutation. Results:,No mutation or Ku80 variant at the RNA level was seen in any patient samples or in the Raji or CCRF-CEM cell lines. In Ku80 expression, 8.8-, 1.9-, and 6.2-fold mean increases were seen in adult, pediatric ALL, and chronic lymphoid malignancies compared with the control. The Ku80 was significantly higher in adult than in pediatric ALL (P = 0.02). The amount of Ku80 expression in ALL was moderately correlated with peripheral white blood cell counts, but not with Ki67 labeling index. High Ku80 expressers (higher than the mean of all patients with ALL) tended to respond poorly to therapy: Only 22% of high Ku80 expressers achieved durable complete remission compared to 62% of low expressers. Conclusions:,Our study suggests that Ku80 might contribute to generally poor prognoses in adult ALL. [source] Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolutionEVOLUTION AND DEVELOPMENT, Issue 1 2005Shigehiro Kuraku Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft-shelled turtle, Pelodiscus sinensis, using microbead-based differential cDNA analysis and real-time reverse transcription-polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid-binding protein-I (CRABP-I), adenomatous polyposis coli down-regulated 1 (APCDD1), and lymphoid enhancer-binding factor-1 (LEF-1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF-1, the nuclear localization of ,-catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co-option of pre-existing genes. [source] In vitro culture of skin-homing T lymphocytes from inflammatory skin diseasesEXPERIMENTAL DERMATOLOGY, Issue 5 2005Karen Bang Abstract:, We, in this study, describe how T lymphocytes in a skin biopsy can proliferate in vitro for up to 3 months by using T-cell growth factors , interleukin-2 (IL-2) and IL-4 yielding approximately 100,160 million T lymphocytes within 1 month. We established cell lines from three tuberculin skin tests, four positive patch tests, 15 of 16 biopsies from atopic dermatitis (AD), 15 of 19 biopsies from mycosis fungoides (MF), 12 of 24 biopsies from psoriasis vulgaris, which was significantly less than AD (P < 0.05), and with a reduced cumulative number of lymphocytes (P < 0.05). Omitting IL-2 and IL-4 led to immediate halt of proliferation. Blood mononuclear cells from patients and biopsies from healthy persons never gave cell lines. All cells were T lymphocytes expressing CD45RO+, HLA-DR+ and CD150. The CD7 expression was significantly increased in cell lines from AD (P < 0.05). T-cell receptor ,-chain studies by using reverse transcription-polymerase chain reaction showed that all T lymphocytes had access to the skin compartment. Single-stranded conformational analysis showed clonally expanded T cells numbering between 40 and 60 clones. After approximately 2 months of growth, the mean CD4+ : CD8+ ratio was for AD 1.20, MF 0.65 and psoriasis 0.85. Patients with AD treated with cyclosporin-A had almost no growth of CD8+ cells in vitro. Our findings indicate a changed homeostasis among skin-homing lymphocytes for in vitro culture. Our culture system of skin-homing T lymphocytes leads to a prominent cellular expansion allowing for a range of studies of in vivo activated skin T lymphocytes. [source] Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp.FEMS MICROBIOLOGY LETTERS, Issue 1 2003PCC 680 Abstract The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S -adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of ,700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome. [source] Nucleoside transporter expression and function in cultured mouse astrocytesGLIA, Issue 1 2005Liang Peng Abstract Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755,760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1,4°C to study diffusional uptake and 1,60-min uptakes at 37°C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na+ -independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na+ -dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine. © 2005 Wiley-Liss, Inc. [source] Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway,HEPATOLOGY, Issue 6 2006Clifford S. Guy The liver plays an increasingly recognized role in the host's immune responses. The direct contribution of hepatocytes as effector cells to local immunity, pathogen containment, and liver disease is not determined. This in vitro study examined whether hepatocytes can eliminate other cells via a CD95 ligand (CD95L or FasL)/CD95 (Fas),mediated mechanism and whether this cytotoxic activity can be modulated by cytokines such as interferon gamma (IFN-,) or tumor necrosis factor alpha (TNF-,). We have found that normal woodchuck and human hepatocytes, both cultured and primary freshly isolated, as well as human HepG2 cells, intrinsically transcribe not only CD95 but also CD95L when examined by reverse transcription-polymerase chain reaction (RT-PCR) assays. The functional competence of CD95L, which was detectable in hepatocytes and HepG2 cells by Western blotting, was confirmed in bioassays by induction of apoptosis of CD95-bearing P815 and LS102.9 cell targets and validated by inhibition of the cell killing with CD95 antagonistic antibody or with a general caspase inhibitor. Furthermore, exposure of cultured hepatocytes to IFN-, or their stable transfection with IFN-, cDNA or TNF-, cDNA increased hepatocyte CD95L/CD95,mediated cell killing. In conclusion, hepatocytes express both CD95L and CD95 and they can induce death of other cells by a CD95L-dependent mechanism. IFN-, and, to a lesser extent, TNF-, can enhance hepatocyte CD95L-mediated cytotoxicity. This suggests that the local cytokine environment may modulate the hepatocyte contribution to liver immunity. (HEPATOLOGY 2006;43:1231,1240.) [source] Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" biliary cancer chemically induced in rat liverHEPATOLOGY, Issue 6 2000Guan-Hua Lai Recently, we observed that Met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is overexpressed in epithelial cells of both early-appearing intestinal metaplastic glands in precancerous hepatic cholangiofibrotic tissue and neoplastic glands in later developed intestinal-type of cholangiocarcinoma originated from the furan rat model of cholangiocarcinogenesis when compared with normal and hyperplastic intrahepatic biliary epithelia. We now show that HGF/SF is also aberrantly expressed in a manner closely paralleling that of its receptor in the neoplastic epithelial cells of furan-induced rat cholangiocarcinomas and in a majority of metaplastic epithelial cells within earlier formed precancerous hepatic cholangiofibrotic tissue. Using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), we further showed specific expression of HGF/SF messenger RNA (mRNA) in a novel rat cholangiocarcinoma epithelial cell line overexpressing Met. This cholangiocarcinoma cell line, termed C611B, was established from tumorigenic cells isolated from a furan-induced transplantable tumor. Moreover, we detected by in situ hybridization strong expression of HGF/SF mRNA transcripts in the cancerous epithelial glands of cholangiocarcinoma developed in recipient rats after in vivo cell transplantation of C611B cells. In contrast, mRNA transcripts and protein immunoreactivity for this cytokine were not detected in hepatocytes and biliary epithelial cells in adult normal rat liver nor in rat hyperplastic intrahepatic biliary epithelium. Our results clearly show that HGF/SF becomes aberrantly expressed in cholangiocarcinoma epithelium and in putative precancerous intestinal metaplastic epithelium induced in the liver of furan-treated rats. [source] DNA demethylation of vascular endothelial growth factor-C is associated with gene expression and its possible involvement of lymphangiogenesis in gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Shunji Matsumura Abstract Previous studies have indicated that lymphangiogenesis in solid tumors is associated with lymphatic metastasis. Overexpression of Vascular endothelial growth factor (VEGF)-C plays a major role in lymphangiogenesis in cancers. In the present study, DNA methylation and expression of the VEGF-C gene was investigated in gastric cancer (GC). Four GC cell lines (MKN-45, MKN-74, HSC-39 and HSC-43) showed no expression of VEGF-C, and the VEGF-C gene was found to be methylated in these cells. In contrast, 7 GC cell lines (MKN-1, MKN-7, MKN-28, TMK-1, KATO-III, SH101-P4 and HSC-44PE) expressed VEGF-C, and the VEGF-C gene was found to be unmethylated in these cell lines. In addition, expression of VEGF-C mRNA was retrieved by treatment with a demethylating agent, Aza-2,-deoxycytidine. In GC tissue samples, bisulfite DNA sequencing analysis revealed that VEGF-C was not methylated in 9 (29.0%) of 31 GC samples, whereas demethylation was not observed in corresponding non-neoplastic mucosa samples. Overexpression of VEGF-C mRNA was observed in 16 (51.6%) of 31 GC samples by quantitative reverse transcription-polymerase chain reaction. Of the 9 GC cases with VEGF-C demethylation, 8 (88.9%) overexpressed VEGF-C. In contrast, of the 22 GC cases without VEGF-C demethylation, 8 (36.4%) overexpressed VEGF-C (p = 0.0155). Furthermore, lymphatic vessel density determined by immunostaining of podoplanin in GC tissues was associated with overexpression of VEGF-C (p < 0.0001). These results suggest that demethylation and activation of the VEGF-C gene is likely involved in lymphangiogenesis in GC. © 2007 Wiley-Liss, Inc. [source] Interleukin-10 expression significantly correlates with minor CD8+ T-cell infiltration and high microvessel density in patients with gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006Teruhisa Sakamoto Abstract We aimed to investigate the relationships between interleukin-10 (IL-10) expression and both the clinicopathological findings and prognoses in patients with gastric cancer and to compare IL-10 expression with microvessel (MV) density and CD8+ T lymphocyte infiltration to evaluate its effects on angiogenesis and immune responses in gastric cancer. IL-10 expression was determined in gastric cancer patients by reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemical procedures. Two of 7 normal gastric tissues showed IL-10 mRNA expression, while its expressions were confirmed in all advanced gastric carcinoma tissues examined (n = 11) by RT-PCR. Immunohistochemical staining demonstrated that IL-10 expression was detected in 52 (47.7%) of 109 cases. There was a close correlation between IL-10 expression and MV density. IL-10 expression inversely correlated with CD8+ T-lymphocyte infiltration. The prognoses of patients whose tumors expressed IL-10 were significantly worse than those of patients whose tumors did not express IL-10. Multivariate analysis indicated IL-10 expression was an independent prognostic factor. IL-10 might be associated with tumor progression by stimulating angiogenesis and suppressing immune responses in gastric cancer. © 2005 Wiley-Liss, Inc. [source] Adrenomedullin regulates expressions of transforming growth factor-,1 and ,1-induced matrix metalloproteinase-2 in hepatic stellate cellsINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2006Yi Wang Summary Adrenomedullin (AM), a peptide isolated from human pheochromocytoma, can be produced and secreted by various types of cells including hepatic stellate cells (HSCs), and its possible role in HSCs is not clear now. In the present study, the interactive regulation between transforming growth factor (TGF)-,1 and AM and the effect of AM on TGF-,1-induced matrix metalloproteinase (MMP)-2 expression in HSCs were investigated. TGF-,1 and AM inhibited gene transcript level mutually (real-time reverse transcription-polymerase chain reaction). AM suppressed the protein expression level of TGF-,1 (Western blot), but TGF-,1 might have no effect on AM secretion level. MMP-2 protein expression in HSCs was increased in response to TGF-,1, and upregulation of MMP-2 expression stimulated with TGF-,1 was suppressed by AM in dose-dependent manner (Western blot). AM decreased the phosphorylation level of extracellular signal-regulated kinase (ERK) in HSCs treated with TGF-,1, and TGF-,1-induced MMP-2 expression was suppressed by adding Mitogen-activated protein Kinase/ERK (MEK) inhibitor U0126 (Western blot). Our results suggest that AM may intervene the activation of HSCs by inhibiting TGF-,1 production and TGF-,1-induced MMP-2 expression; AM may suppress the upregulation of MMP-2 expression induced by TGF-,1 partially through ERK pathway. [source] Establishment and characterization of androgen-independent human prostate cancer cell lines, LN-REC4 and LNCaP-SF, from LNCaPINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2007Yoichi Iwasa Aim: To investigate the mechanisms of androgen-independent growth in prostate cancer (PCa), we established two PCa cell lines, LN-REC4 and LNCaP-SF, from the androgen-dependent PCa cell line, LNCaP. Materials and methods: LN-Pre and LN-REC4 cells were generated from LNCaP tumors grown on intact and castrated severe combined immunodeficient (SCID) mouse, respectively. After we cultured LNCaP cells under a steroid-free conditions for 6 months in vitro, LNCaP-SF cells were established. To show the character of LN-REC4 and LNCaP-SF cells, androgen sensitivity was investigated through examination of growth rate, and prostate-specific antigen (PSA), androgen receptor (AR), p21, p27, and cyclin D1 expression were examined by reverse transcription-polymerase chain reaction (RT-PCR). Angiogenesis assay in vitro was carried out using conditioned medium. To examine the expression level of vascular endothelial growth factor (VEGF), RT-PCR and enzyme-linked immunosorbent assay were also done. Results and conclusions: LN-REC4 cells proliferated better than LNCaP cells in castrated mice and did well irrespective of castration, although responsiveness for androgen of LN-REC4 cells attenuated less than that of LNCaP cells in vitro. LNCaP-SF cells in castrated mice proliferated more rapidly than in normal mice. The PSA expression in LNCaP-SF cells was still induced by androgen. Expression of AR, p21, p27 and cyclin D1 were not changed in LN-REC4 and LNCaP-SF cells. Angiogenesis assay showed that both cells stimulated angiogenesis. LN-REC4 induced VEGF more than LNCaP and LN-Pre cells. However, expression of VEGF per cell in LNCaP-SF was lower than LNCaP cells, suggesting that other factors might be involved in angiogenesis. These cell lines might be a useful tool for researching androgen-independent growth and treatments of recurred PCa. [source] Global analysis of gene expression profiles in ileum in a rat bladder augmentation model using cDNA microarraysINTERNATIONAL JOURNAL OF UROLOGY, Issue 11 2004HIDEAKI MIYAKE Abstract Background: The objective of the present study was to globally characterize the changes in the gene expression profile in the ileum after long-term urine exposure in a rat ileal augmented bladder model using cDNA microarrays. Methods: Bladder augmentation using the ileum was performed in female 8-week-old rats. The ileal epithelia used for bladder augmentation were harvested 1 and 3 months postoperatively and changes in the gene expression in these tissues were compared with that of intact ileal epithelia from sham-operated rats using cDNA microarrays consisting of 1176 rat genes. Results: Marked changes in gene expression in the ileum used for bladder augmentation were observed for 30 genes (16 up-regulated and 14 down-regulated genes). The differentially expressed genes include those associated with signal transduction, cell adhesion and stress response. Subsequent evaluation of changes in two randomly selected genes from the 30 differentially expressed genes by semiquantitative reverse transcription-polymerase chain reaction demonstrated the reliability of the present cDNA microarray analyses. Conclusion: The present experiments identified an extensive list of genes differentially expressed in the ileum after bladder augmentation, providing valuable information for the pathophysiological assessment of patients who undergo urinary reconstruction and representing a source of novel targets for treating complications after urinary diversion. [source] Vasoactive intestinal polypeptide immunoreactivity in the human cerebellum: qualitative and quantitative analysesJOURNAL OF ANATOMY, Issue 3 2009Vincenzo Benagiano Abstract Although autoradiographic, reverse transcription-polymerase chain reaction and immunohistochemical studies have demonstrated receptors for vasoactive intestinal polypeptide (VIP) in the cerebellum of various species, immunohistochemistry has never shown immunoreactivity for VIP within cerebellar neuronal bodies and processes. The present study aimed to ascertain whether VIP immunoreactivity really does exist in the human cerebellum by making a systematic analysis of samples removed post-mortem from all of the cerebellar lobes. The study was carried out using light microscopy immunohistochemical techniques based on a set of four different antibodies (three polyclonal and one monoclonal) against VIP, carefully selected on the basis of control tests performed on human colon. All of the antibodies used showed VIP-immunoreactive neuronal bodies and processes distributed in the cerebellar cortex and subjacent white matter of all of the cerebellum lobes, having similar qualitative patterns of distribution. Immunoreactive neurons included subpopulations of the main neuron types of the cortex. Statistical analysis of the quantitative data on the VIP immunoreactivity revealed by the different antibodies in the different cerebellar lobes did not demonstrate any significant differences. In conclusion, using four different anti-VIP antibodies, the first evidence of VIP immunoreactivity is herein supplied in the human post-mortem cerebellum, with similar qualitative/quantitative patterns of distribution among the different cerebellum lobes. Owing to the function performed by VIP as a neurotransmitter/neuromodulator, it is a candidate for a role in intrinsic and extrinsic (projective) circuits of the cerebellum, in agreement with previous demonstrations of receptors for VIP in the cerebellar cortex and nuclei. As VIP signalling pathways are implicated in the regulation of cognitive and psychic functions, cerebral blood flow and metabolism, processes of histomorphogenesis, differentiation and outgrowth of nervous tissues, the results of this study could be applied to clinical neurology and psychiatry, opening new perspectives for the interpretation of neurodevelopment disorders and development of new therapeutic strategies in cerebellar diseases. [source] Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virusJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008Z. Liu Abstract Aim:, Spring viraemia of carp virus (SVCV) is the causative agent of SVC disease. The main aim of our study was to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and effective detection of SVCV. Methods and Results:, A set of four specific primers, two outer and two inner primers were designed based on the SVCV M gene for RT-LAMP assay. The sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification conditions (64°C, 60 min). The results showed that the assay has a high specificity and the detection limit was 80 copies using 10-fold series dilutions of SVCV RNA, 10 times more sensitive than nest reverse transcription-polymerase chain reaction. In the detection of 472 fish samples, this assay showed excellent agreement with the standard virus isolation method (, = 0·807). Conclusions:, A sensitive and specific RT-LAMP assay was successfully developed to monitor and detect SVCV. Significance and Impact of the Study:, This work provides a robust method for evaluating the risk of SVCV. Given the advantages of LAMP in the detection of SVCV, this method can be applied to diagnose other viruses, which pose serious threats to the aquaculture industry. [source] Metal ion enhancement of fungal growth, gene expression and aflatoxin synthesis in Aspergillus flavus: RT-PCR characterizationJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2003R. Cuero Abstract Aims: To determine the effect of mineral ions (e.g., Zn2+, Cu2+, and Fe2+) on the enhancement of fungal growth, total RNA, aflatoxin pathway gene expression, and production of aflatoxin and its precursor O -methylsterigmatocystin (OMST). Methods and Results: The influence of the metal ions, as a single or mixed treatments, was observed in submerged cultures of toxigenic Aspergillus flavus through changes in the fungal RNA or aflatoxin pathway gene (omtA) by reverse transcription-polymerase chain reaction (RT-PCR) levels, and also in fungal dry-weight accumulation, aflatoxin and OMST production. Conclusions: The ion treatments induced changes of fungal total RNA, mRNA levels, associate fungal growth, biosynthesis of aflatoxin and OMST, and enhanced expression of RT-PCR. Significance and Impact of Study: Demonstrates at the cellular and molecular level, the significant effects of metal ions on both fungal growth and production of aflatoxin. [source] Cis-Acting Intronic Elements That Regulate Cartilage-Specific Alternative Splicing of the Type II Collagen (Col2) Pre-mRNA Lie at or Near Splice Site Junction Sequences Flanking Exon 2 of the Gene,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2003Takayuki Nishiyama Abstract Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. Introduction: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. Materials and Methods: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. Results: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14,21 days) with insulin. Conclusion: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA. [source] Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003DC Simes Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source] Alendronate Interacts With the Inhibitory Effect of 1,25(OH)2D3 on Parathyroid Hormone-Related Protein Expression In Human Osteoblastic Cells,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2003L Gómez-García Abstract The bisphosphonate alendronate is a potent inhibitor of bone resorption by its direct action on osteoclasts. In addition, there is some data suggesting that alendronate could also inhibit bone resorption indirectly by interacting with osteoblasts. Parathyroid hormone-related protein (PTHrP) produced by osteoblasts and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are regulators of bone remodeling, which have interrelated actions in these cells. In this study, we assessed whether alendronate can affect PTHrP expression in the presence or absence of 1,25(OH)2D3 in human primary osteoblastic (hOB) cells from trabecular bone. Cell total RNA was isolated, and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out using human PTHrP-specific primers. PTHrP in the hOB cell-conditioned medium was analyzed by a specific immunoradiometric assay. We found that PTHrP mRNA and secreted PTHrP were maximally inhibited by 10,8 -10,6 M of 1,25(OH)2D3 treatment within 8,72 h in hOB cells. Alendronate (10,14 -10,8 M) modified neither PTHrP mRNA nor PTHrP secretion, although it consistently abrogated the decrease in PTHrP production induced by 1,25(OH)2D3 in these cells. On the other hand, alendronate within the same dose range did not affect either the vitamin D receptor (VDR) mRNA or osteocalcin secretion, with or without 1,25(OH)2D3, in hOB cells. The inhibitory effect of alendronate on the 1,25(OH)2D3 -induced decrease in PTHrP in these cells was mimicked by the calcium ionophore A23187 (5 × 10,6 M), while it was eliminated by 5 × 10,5 M of nifedipine. Furthermore, although alendronate alone failed to affect [Ca2+]i in these cells, it stimulated [Ca2+]i after pretreatment of hOB cells with 10,8 M of 1,25(OH)2D3, an effect that was abolished by 5 × 10,5 M of nifedipine. These results show that alendronate disrupts the modulatory effect of 1,25(OH)2D3 on PTHrP production in hOB cells. Our findings indicate that an increase in calcium influx appears to be involved in the mechanism mediating this effect of alendronate. [source] Bepridil Reverses Atrial Electrical Remodeling and L-Type Calcium Channel Downregulation in a Canine Model of Persistent Atrial TachycardiaJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 7 2007KUNIHIRO NISHIDA M.D. Introduction: This study tested whether bepridil, a multichannel blocker, would reverse electrical remodeling induced by persistent atrial tachycardia. Methods and Results: Fourteen dogs were subjected to rapid atrial pacing at 400 bpm for 6 weeks after atrioventricular block was created to control the ventricular rate. During the study period, seven dogs were given placebo for 6 weeks (Control group), and seven were given placebo for 3 weeks, followed by 3 weeks of bepridil (10 mg/kg/day, Bepridil group). The atrial effective refractory period (ERP) and the inducibility and duration of atrial fibrillation (AF) were determined on a weekly basis. After 6 weeks, expression of L-type calcium channel ,1C messenger ribonucleic acid (mRNA) was quantified by real-time reverse transcription-polymerase chain reaction. In the Control group, ERP was shortened and the inducibility and duration of AF increased through the 6-week period. In the Bepridil group, the same changes occurred during the first 3 weeks, but were gradually reversed with bepridil. After 6 weeks, ERP was longer, AF inducibility was lower, and AF duration was shorter in Bepridil group than in the Control group. Expression of ,1C mRNA was decreased by 64% in the Control group (P < 0.05 vs sham), but in the Bepridil group, it was not different compared with the sham dogs. As a whole group of dogs, ERP was positively correlated with ,1C mRNA expression. Conclusion: Bepridil reverses the electrophysiological consequences of atrial remodeling to some extent and L-type calcium channel downregulation in a canine model of atrial tachycardia. [source] DNA methylation and histone modification regulate silencing of OPG during tumor progression,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Tung-Ying Lu Abstract The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression. J. Cell. Biochem. 108: 315,325, 2009. © 2009 Wiley-Liss, Inc. [source] ,-cryptoxanthin stimulates cell differentiation and mineralization in osteoblastic MC3T3-E1 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Satoshi Uchiyama Abstract The effect of ,-cryptoxanthin, a kind of carotenoid, on cell differentiation and mineralization in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in a minimum essential medium containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or ,-cryptoxanthin (10,8 to 10,6 M) without FBS. Cells were cultured for 3 to 21 days. Gene expression in osteoblastic cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with ,-cryptoxanthin (10,7 or 10,6 M) for 3 days caused a significant increase in Runx2 type 1, Runx2 type 2, ,1 (I) collagen, and alkaline phosphatase mRNA levels in osteoblastic cells. These increases were completely blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-,- D -ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. Meanwhile, vitamin A (10,6 M) did not have a significant effect on Runx2 type 1 mRNA expression in the cells. The effect of ,-cryptoxanthin (10,6 M) in stimulating Runx2 type 1 and ,1 (I) collagen mRNA levels, protein content, and alkaline phosphatase activity in the cells was also seen in the presence of vitamin A (10,6 M), suggesting that the mode of ,-cryptoxanthin action differs from that of vitamin A. Prolonged culture with ,-cryptoxanthin (10,6 M) for 3 to 21 days caused a significant increase in cell number, deoxyribonucleic acid (DNA) content, protein content, and alkaline phosphatase activity in osteoblastic cells, suggesting that ,-cryptoxanthin stimulates cell proliferation and differentiation. Moreover, culture with ,-cryptoxanthin (10,7 or 10,6 M) for 5 to 21 days caused a remarkable increase in mineralization. This study demonstrates that ,-cryptoxanthin has a stimulatory effect on cell differentiation and mineralization due to enhancing gene expression of proteins, which involve in bone formation in osteoblastic MC3T3-E1 cells. © 2005 Wiley-Liss, Inc. [source] Differential cytokine expression by human dendritic cells in response to different Porphyromonas gingivalis capsular serotypesJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 10 2009Rolando Vernal Abstract Aim: Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. Materials and Methods: Using different multiplicity of infection (MOI) of the encapsulated strains K1,K6 and the non-encapsulated K, strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1,, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)- ,, tumour necrosis factor (TNF)- ,, and TNF- , in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. Results: All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1,, IL-6, IL-12p35, IL-12p40, and IFN- , and at lower MOI than DCs stimulated with the other strains. Conclusions: These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression. [source] Effects of antisense oligodeoxynucleotide to type I collagen gene on hypertrophic scars in the transplanted nude mouse modelJOURNAL OF CUTANEOUS PATHOLOGY, Issue 11 2009Julin Xie Background:, Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression. We had previously proved that the antisense DNA to type I collagen could effectively inhibit the synthesis of collagen type I in cultured hypertrophic scar fibroblasts, suggesting a potential role in anti-scarring, but there are no published reports of its effect on scar in the transplanted nude mouse model. Aims:, To investigate the effects of antisense oligodeoxynucleotide (ASODN) to type I collagen gene on hypertrophic scars in the transplanted nude mouse model and clarify the potential of ASODN for the treatment of scars. Methods:, The nude mouse model of hypertrophic scar was created and subjected to daily injections with ASODN and LipofectamineÔ for 2 ,4 or 6 weeks. We then examined the scars for changes in histopathological characteristics. The effects of ASODN on type I collagen gene expression were examined by reverse transcription-polymerase chain reaction and Western blots. Results:, The ASODN could remarkably alleviate the scar in the nude mouse model and consistently inhibit type I collagen gene expression at both the mRNA and protein levels. Conclusion:, ASODN was effective in downregulating type I collagen gene expression and could prove to be useful in the treatment of scars. [source] | ||||||||||||||||||||||||||||||||||||||||