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Reverse Transcription (reverse + transcription)
Kinds of Reverse Transcription Terms modified by Reverse Transcription Selected AbstractsAnalysis of Sir2E in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpressionDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2008Takahiro Katayama It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A,D) showing sequence similarity to human homologues of Sir2 (SIRT1,3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription,polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum. [source] Cloning and characterization of cDNA for syndecan core protein in sea urchin embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000Kazuo Tomita The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source] RET rearrangements and BRAF mutation in undifferentiated thyroid carcinomas having papillary carcinoma componentsHISTOPATHOLOGY, Issue 3 2010Kunio Mochizuki Mochizuki K, Kondo T, Nakazawa T, Iwashina M, Kawasaki T, Nakamura N, Yamane T, Murata S-i, Ito K, Kameyama K, Kobayashi M & Katoh R (2010) Histopathology,57, 444,450 RET rearrangements and BRAF mutation in undifferentiated thyroid carcinomas having papillary carcinoma components Aims:, To elucidate the genetic background of anaplastic transformation, RET rearrangements and BRAF mutation were studied in composite undifferentiated carcinomas (UCs) of the thyroid, which are UCs having papillary carcinoma (PC) components. Methods and results:, Reverse transcription,polymerase chain reaction (RT,PCR) was performed for RET rearrangements and PCR for BRAF mutation in UC and PC components that were microdissected separately from seven composite UCs. Forty-two thyroid cancers with single component histology (14 UCs and 28 PCs) were also studied in the same manner. RET/PTC1 was undetectable in both components from all seven composite UCs, and RET/PTC3 was identified in both components of one composite UC. BRAF mutation was identified in both components from three composite UCs and only in the PC components from two composite UCs. In contrast, in thyroid carcinomas with single component histology, RET/PTC1 was detected in 11% of PCs and in none of the UCs, and RET/PTC3 was not found in any of the tumours studied. BRAF mutation was identified in 82% of PCs and in 21% of UCs. Conclusions:, The high frequency of BRAF mutation and the absence of RET rearrangements in UC components from composite UCs supports the hypothesis that UCs may actually represent progressive malignant degeneration of a BRAF -mutated, well-differentiated thyroid carcinoma. [source] Cytokine-mediated control of lipopolysaccharide-induced activation of small intestinal epithelial cellsIMMUNOLOGY, Issue 3 2007Michael Lotz Summary Cytokines with anti-inflammatory properties have been implicated in the prevention of inappropriate immune activation by commensal bacteria in the intestinal tract. Here, we analysed receptor expression, cellular signalling, and the inhibitory activity of interleukin (IL)-4, -10, -11, and -13 as well as of transforming growth factor-, on lipopolysaccharide-mediated small intestinal epithelial cell activation. Only IL-4 and IL-13 had a significant inhibitory effect on chemokine secretion and nitric oxide (NO) production in differentiated and polarized cells. Reverse transcription,polymerase chain reaction of primary intestinal epithelial cells obtained by laser-microdissection confirmed expression of the type II IL-4 receptor consisting of the IL-4 receptor , and the IL-13 receptor ,1. Also, IL-4 or IL-13 led to rapid signal transducer and activator of transcription 6 phosphorylation, diminished inducible NO synthase expression, and enhanced the antagonistic arginase 1 activity. In conclusion, cytokines such as IL-4 and IL-13 affect intestinal epithelial cells and exhibit a modulating activity on Toll-like receptor-4-mediated epithelial cell activation. [source] cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparumINSECT SCIENCE, Issue 3 2009Jia-Ying Zhu Abstract, This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length cDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein (1.42% methionine and 18.82% aromatic amino acids). Reverse transcription , polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum. [source] Expression of matrix metalloproteinase-1, matrix metalloproteinase-2 and extracellular metalloproteinase inducer in human periodontal ligament cells stimulated with interleukin-1betaJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2009J. Xiang Background and Objectives: Matrix metalloproteinases (MMPs), produced by both infiltrating and resident cells of the periodontium, play important roles in physiologic and pathologic events. Both interleukin-1beta and extracellular MMP inducer can stimulate the expression of MMPs, which in turn leads to breakdown of the periodontium. However, it is currently unknown whether interleukin-1beta up-regulates MMPs through stimulating the expression of extracellular MMP inducer. The aims of this study were to investigate the effect of interleukin-1beta on the expression of MMP-1, MMP-2 and extracellular MMP inducer in human periodontal ligament cells and to evaluate whether the regulation of MMP-1 and MMP-2 by this cytokine occurred through an effect on extracellular MMP inducer expression. Material and Methods: Cultured human periodontal ligament cells were treated with varying concentrations (0.01,10 ng/mL) of interleukin-1beta at for 6, 12 and 24 h. Reverse transcription,polymerase chain reaction, enzyme-linked immunosorbent assay, gelatin zymography and western blotting were performed to measure the mRNA and protein levels of MMP-1, MMP-2 and extracellular MMP inducer. Results: Basal levels of mRNA and protein for MMP-1, MMP-2 and extracellular MMP inducer were detected in untreated human periodontal ligament cells. Interleukin-1beta significantly up-regulated the expression of MMP-1 and MMP-2 mRNA and protein (p < 0.05); however, the levels of mRNA and protein for extracellular MMP inducer were not significantly different (p > 0.05). In the culture medium, the concentration of MMP-1 was also increased significantly, but the concentration of MMP-1 was not related to the concentration of extracellular MMP inducer (R2 = 0.2538, p > 0.05). Conclusion: Interleukin-1beta up-regulated the levels of MMP-1 and MMP-2, but it did not alter the expression of extracellular MMP inducer. Expression of MMP-1 and MMP-2 might be elevated by interleukin-1beta and extracellular MMP inducer via two different signal pathways. [source] Decreased expressions of thrombospondin 2 in cyclosporin A-induced gingival overgrowthJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2004Jeong Tae Koh Objectives:, Cyclosporin A (CsA) is known to elicit fibrous gingival overgrowth with changes of blood vessel profiles. In this study, we examined the expression of several angiogenic and angiostatic genes during the development of CsA-induced gingival overgrowth. Methods:, For the development of gingival overgrowth, Sprague-Dawley rats received subcutaneous injections of CsA in daily doses of 5, 10, 15 mg/kg body weight for 6 weeks, and another group received 10 mg/kg of CsA for 3, 6, and 12 weeks. Human gingival tissues were obtained from three CsA-treated patients following the gingivectomy procedure and from three healthy patients following the crown-lengthening procedure as a control. Gingival fibroblasts were isolated from the healthy gingival tissues of the rat or the human, and cultured with 250,1000 ng/ml of CsA. Results:, Reverse transcription,polymerase chain reaction (RT,PCR) analyses showed that expressions of some angiogenic genes such as angiopoietin 1, basic fibroblast growth factor, and vascular endothelial growth factor, and angiostatic genes such as angiopoietin 2, brain-specific angiogenesis inhibitor 1 and 2, and thrombospondin 1 were not changed significantly in both gingival tissues and cultured fibroblast cells under the CsA treatments. However, expression of thrombospondin 2 (TSP2) decreased dose- and time-dependently in rat and human gingival tissues. Western blot analyses showed that the expression of TSP2 protein was dose-dependently reduced by the CsA treatments in human cultured gingival fibroblasts. Conclusions:, These results indicate that the decrease in angiostatic TSP2 expression may be attributed to the CsA-induced gingival vascularization rather than to the increased expression of angiogenic genes. It suggests that TSP2 is involved in the development of CsA-induced gingival overgrowth with the gingival vascularization. [source] Comparative study between the effect of the peroxisome proliferator activated receptor-, ligands fenofibrate and n-3 polyunsaturated fatty acids on activation of 5,-AMP-activated protein kinase-,1 in high-fat fed ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2009Tarek M. Kamal Motawi Abstract Objectives Obesity is a risk factor for type 2 diabetes mellitus. It results from an energy imbalance in which energy intake exceeds energy expenditure. The cellular fuel gauge 5,-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of one catalytic subunit (,) and two non-catalytic subunits (, and ,), and approximately equal levels of ,1 and ,2 complexes are present in the liver. AMPK regulates metabolic pathways in response to metabolic stress and in particular ATP depletion to switch on energy-producing catabolic pathways such as ,-oxidation of fatty acids and switch off energy-depleting processes such as synthesis of fatty acid and cholesterol. A high-fat diet alters AMPK-,1 gene expression in the liver and skeletal muscle of rats and results in body weight gain and hyperglycaemia. The aim of this study was to investigate and compare the potential effects of peroxisome proliferator-activated receptor (PPAR)-, agonists fenofibrate and n-3 polyunsaturated fatty acids (PUFAs) in modulation of AMPK-,1 activity in liver and skeletal muscle of high-fat diet fed rats. Methods Reverse transcription,polymerase chain reaction was used for determination of AMPK-,1 in liver and soleus muscle and both PPAR-, and CPT-1 in hepatic tissues. Serum, total cholesterol, triacylglycerol, fatty acid and fasting blood glucose were determined colorimetrically. Key findings Both PPAR-, agonists, fenofibrate and n-3 PUFA, increased the mRNA expression of AMPK-,1 activity in liver and skeletal muscle of obese diabetic rats. Fenofibrate was superior in its activation of hepatic mRNA expression of AMPK-, 1 to exert more lipolytic effect and body weight reduction, as estimated through the decrease of triacylglycerol output and serum levels of fatty acid on the one hand and the increase in CPT-1 mRNA expression, the key enzyme in ,-oxidation of fatty acid, on the other hand. n-3 PUFA activated AMPK-,1 mRNA expression in skeletal muscle much more than fenofibrate to reveal more hypoglycaemic effect. Conclusions The PPAR-, agonists fenofibrate and n-3 PUFA could efficiently activate AMPK-,1 mRNA expression in liver and skeletal muscle to exert body weight reduction and hypoglycaemic effect, respectively. [source] The expression of SIAH1 is downregulated and associated with Bim and apoptosis in human breast cancer tissues and cellsMOLECULAR CARCINOGENESIS, Issue 5 2010Yuan-Yuan Wen Abstract Seven in absentia homolog1 (SIAH1) was reported as a tumor suppressor and played an important role in regulating cell apoptosis. However, its effects on the breast carcinogenesis remain unclear. In this study, our aims were to examine the relationship between SIAH1 and Bcl-2-interacting mediator of cell death (Bim) and to explore the effects of SIAH1 on the breast carcinogenesis. Immunohistochemical analysis in 231 cases of breast tissues showed that the expression of SIAH1 and Bim were significantly decreased in the breast carcinogenesis. Moreover, SIAH1 expression was significantly correlated with Bim. Both SIAH1 and Bim expression were significantly higher in well to moderately differentiated and in early-stage breast cancer. Reverse transcription (RT)-polymerase chain reaction (PCR) and Western blot analysis in paired breast cancer tissues and breast cell lines found that the expression of SIAH1 was lower in the breast cancer tissues and cell lines. SIAH1 inducing apoptosis of the breast cancer cells was dependent on Bim. However, SIAH1 inhibiting invasion of the breast cancer cells was independent of Bim. The increase of the function of SIAH1 to upregulate the expression of Bim may play an important role in the progression of breast cancer. Restoration of the function of SIAH1 may be a new therapeutic target of human breast cancer. İ 2010 Wiley-Liss, Inc. [source] EmtA, a rRNA methyltransferase conferring high-level evernimicin resistanceMOLECULAR MICROBIOLOGY, Issue 6 2001Paul A. Mann Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes. [source] Expression pattern of the maternal factor zygote arrest 1 (Zar1) in bovine tissues, oocytes, and embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tiziana A.L. Brevini Abstract Zygote arrest 1 (Zar1) is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse. In this species, Zar1 expression is strictly limited to the oocyte, the zygote and, at a lower level, the 2-cell embryo. Aim of the present study was to analyze the presence and the expression pattern of the Zar1 ortholog in bovine tissues and embryos. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis was performed in a panel of bovine tissues, in oocytes and pre-implantation in vitro produced embryos. The results demonstrated that a Zar1 ortholog is present in cattle. In the adult, the gene is expressed in ovary, testis, muscle, and myocardium. The gene is also expressed in the oocyte, the zygote, and in all the stages of embryonic development until blastocyst formation. A semi-quantitative RT-PCR analysis revealed that Zar1 levels are constant through in vitro development with the exception of the 4-cell stage, when a significant increase is observed. The exposure of fertilized oocytes to the RNA polymerase II inhibitor alpha-amanitin was able to suppress this Zar1 increase indicating that transcription of this gene occurs at the 4-cell stage. Zar1 is conserved in cattle but has an expression pattern different from the mouse. In particular, Zar1 expression in the adult is not limited to the ovary and in the embryo is expressed well beyond the oocyte to embryo transition. Moreover, the identification of Zar1 transcription at the 4-cell stage represents the first characterization of one of the genes expressed in cattle embryos before the major onset of embryonic transcription. Mol. Reprod. Dev. 69: 375,380, 2004. İ 2004 Wiley-Liss, Inc. [source] CD97, CD95 and Fas-L clearly discriminate between chronic pancreatitis and pancreatic ductal adenocarcinoma in perioperative evaluation of cryocut sectionsPATHOLOGY INTERNATIONAL, Issue 2 2002Carsten Boltze It is a major problem to distinguish between pancreatitis and pancreatic adenocarcinoma when it comes to the perioperative evaluation of pancreatic cryocut sections. In this respect, pathologists are showing a steadily growing interest in the potential application of apoptotic and dedifferentiation factors as diagnostic and prognostic markers. This study investigated the mRNA and protein expression of CD97, CD95 and Fas-L in snap-frozen material obtained from human pancreatic ductal adenocarcinomas (PDC; n = 50), tissues from pancreatitis (PT; n = 40) and normal pancreatic tissues (PN; n = 36). Reverse transcription,polymerase chain reaction (RT,PCR) analysis revealed that CD97, CD95 and Fas-L mRNA were expressed on a similarly high level in all tissues. In contrast, short time immunohistochemical evaluation showed that the CD95 protein was strongly expressed in PT and PN, but not in PDC. Fas-L protein was expressed strongly in PDC, whereas only weak or no expression was noted in PT or PN. CD97 protein expression was detected only in PT and in poorly differentiated PDC. Our data demonstrate that CD97, CD95 and Fas-L can be used as additional markers to distinguish between pancreatitis and pancreatic duct cell carcinoma in cryocut sections. [source] Bizarre parosteal osteochondromatous proliferation (Nora's lesion) of the footPATHOLOGY INTERNATIONAL, Issue 10 2001Hisashi Horiguchi A 22-year-old man presented with a growing lump on the fifth metatarsal of the right foot. Radiographically, the lesion was a calcified mass stuck on to the bone. The T2 -weighted magnetic resonance images showed heterogeneity in intensity. A tumor was suspected and an excisional biopsy was done. The lesion was composed of a cartilaginous cap and bone tissue. Histological examination revealed characteristic features of bizarre parosteal osteochondromatous proliferation (BPOP), such as hypercellularity, a blue tinctorial quality in the osteocartilaginous interfaces, and a scattering of binucleated or bizarre enlarged chondrocytes. Immunohistochemically, basic fibroblast growth factor was expressed in nearly all chondrocytes within the cartilaginous cap, while vascular endothelial growth factor was expressed only in enlarged chondrocytes near the osteocartilaginous interfaces. Reverse transcription,polymerase chain reaction detected chondromodulin-I transcripts in the tissue of the cartilaginous cap. These findings indicate that the processes occurring in BPOP are similar to those occurring in endochondral ossification in the growth plate, and they support the concept that BPOP is a reparative process. BPOP is a rare tumorous lesion of the bone and is occasionally confused with other benign or malignant conditions. Thus, it is important to consider the clinical, radiographical and the gross histological features of the lesion when making a diagnosis. [source] C117T variant in the SMN1 gene found in the Japanese populationPEDIATRICS INTERNATIONAL, Issue 1 2007AHMAD HAMIM SADEWA Abstract Background: The SMN genes are closely related to the development of spinal muscular atrophy (SMA); mutated SMN1 causes SMA and functional SMN2 modifies the severity of SMA. SMN1 and SMN2 are almost identical, being distinguished by only five base pair substitutions located at the 3'-end of the genes. Recently, a synonymous DNA variant, C117T, has been identified at the first codon of SMN2 exon 2a in the Caucasian population. It is still a question whether the variant is specific to the Caucasian population, and whether it is found only in SMN2. In order to address these questions, Japanese populations were screened for the presence of C117T in the SMN genes. Methods: To detect the C117T variant in a Japanese population, polymerase chain reaction,restriction fragment length polymorphism was performed in 33 SMA patients homozygous for SMN1 deletion and 106 control individuals. Reverse transcription,polymerase chain reaction (RT-PCR) was performed to clarify whether the variant affects the splicing process of the SMN1 gene. Results: The C117T variant was found in one out of 33 Japanese SMA patients (3.0%) and in seven out of 106 Japanese control individuals (6.6%). There was no significant difference between frequencies in the present data and those reported from the Caucasian population. Notably, the C117T variant was also detected in the SMN1 gene; a control individual with homozygous SMN2 deletion was found to have the variant on one of the SMN1 genes. RT-PCR indicated that this variant of the SMN1 gene was normally transcribed and did not affect the splicing process in this individual. Conclusions: The C117T variant was found not only in the Caucasian population, but also in the Japanese population. In addition, the variant was not specific to SMN2: it was also found in SMN1. RT-PCR indicated that the variant did not affect the splicing process. [source] Vestibular Schwannoma Quantitative Polymerase Chain Reaction Expression of Estrogen and Progesterone Receptors,THE LARYNGOSCOPE, Issue 8 2008Andrew K. Patel MD Abstract Objectives/Hypothesis: Determine the role of estrogen receptor (ER) and progesterone receptor (PR) expression in sporadic and neurofibromatosis 2 (NF2)-related vestibular schwannomas (VS). Growth and proliferation signaling in human VS tumorigenesis may play a key role in molecular therapeutic targeting. VS carry mutations of the NF2 gene encoding the tumor suppressor, merlin, which interacts with ErbB2 in Schwann cells, implicating ErbB receptors in VS tumorigenesis. ErbB receptor family members are overexpressed or constitutively activated in many human tumors, and are effective therapeutic targets in some human cancers. VS occur more frequently in women and are larger, more vascular, and demonstrate increased growth rates during pregnancy. ER and PR may play a role in ErbB pathway activation and VS progression. Study Design: Quantitative real-time polymerase chain reaction (qRT-PCR) for ER and PR messenger RNA was performed using greater auricular and vestibular nerve controls (n = 8), sporadic VS (n = 23), and NF2-related VS (n = 16) tissues. Methods: The qRT-PCR data were normalized with standardization to a single constitutively expressed control gene, human cyclophylin. Results: Reverse transcription of messenger RNA from control and tumor specimens followed by RT Q-PCR demonstrated differences in ER and PR gene expression between sporadic and NF2-related VS. Conclusions: ER and PR expression in VS might have implications for development of a VS-specific drug delivery system using antihormone and ErbB pathway small molecule inhibitors, due to crosstalk between these receptors. These signals may be critical for re-establishing ErbB-mediated cell density dependent growth inhibition. [source] Effect of Osteogenic Induction on the in Vitro Differentiation of Human Embryonic Stem Cells Cocultured With Periodontal Ligament FibroblastsARTIFICIAL ORGANS, Issue 11 2007Bülend Inanç Abstract:, Osteogenesis is one of the principal components of periodontal tissue development as well as regeneration. As pluripotent cells with unlimited proliferative potential and differentiation ability to all germ layer representatives, embryonic stem cells also hold the promise to become a cell source in bone tissue engineering. Our aim was to investigate osteogenic differentiation potential of human embryonic stem cells (hESCs) under the inductive influence of human periodontal ligament fibroblast (hPDLF) monolayers. After being expanded and characterized morphologically and immunohistochemically, hESCs (HUES-9) were cocultured with hPDLFs for 28 days. Two groups were established: (i) osteogenic induction group with ascorbic acid, ,-glycerophosphate, and dexamethasone containing hESC differentiation medium; and (ii) spontaneous differentiation group cultured in hESC differentiation medium. Morphological shift in cells was analyzed under an inverted microscope, and immunohistochemistry was performed on fixed specimens at days 1 and 28 using antibodies against alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein (BSP), and osteocalcin (OSC). Reverse transcription,polymerase chain reaction was utilized for the detection of octameric binding protein-4, BSP, and OSC expression at mRNA level. Mineralization was assessed using alizarin red, and the surface topology shift in colonies was demonstrated with scanning electron microscopy. Results indicate the feasibility of osteogenic differentiation of hESCs in coculture, and suggest a role of periodontal ligament fibroblasts in their differentiation patterns. Advances in the field could allow for potential utilization of hESCs in periodontal tissue engineering applications involving regeneration of bone in periodontal compartment lost as a result of destructive periodontal diseases. [source] Additional reverse transcription,polymerase chain reaction of peripheral slices is not superior to analysis of the central slice in sentinel lymph nodes from melanoma patientsBRITISH JOURNAL OF DERMATOLOGY, Issue 3 2004H-J. Blaheta Summary Background The status of the sentinel lymph node (SLN) is an important prognostic factor in patients with cutaneous melanoma. Reverse transcription,polymerase chain reaction (RT,PCR) has been used as a sensitive means of detecting tumour cells in SLNs. Objectives To determine whether RT,PCR analysis of the SLN using both the central and the peripheral slices is more sensitive than molecular analysis of the central slice only. Methods Eighty-three SLNs from 59 patients with primary cutaneous melanoma were identified by SLN mapping. All SLNs were bisected along their longitudinal axis to produce two equal halves. One half was used for histology and immunohistochemistry, and the other was analysed by RT,PCR for tyrosinase and MelanA. Parallel to the longitudinal axis, one central slice (approximately 2 mm in thickness) was cut manually. This central slice was used for our standard RT,PCR protocol. In the current study, up to eight additional peripheral slices (each approximately 2 mm in thickness) were cut parallel to the existing cut surface. These peripheral slices were analysed by additional RT,PCR. Results Standard RT,PCR of the central slice yielded positive results in 34 of 59 patients (57%). Additional RT,PCR of peripheral slices demonstrated positive findings in six additional patients (10%) who were initially negative by standard RT,PCR of the central slice. In detail, seven of those 34 patients positive by standard RT,PCR of the central slice had positive histological findings. In each of these seven patients, RT,PCR was positive both in the central slice as well as in the peripheral slices. The remaining 27 patients with positive RT,PCR results of the central slice showed negative histological findings. Only nine (33%) of these 27 patients had a positive RT,PCR also in the peripheral slices. Finally, all 25 patients with negative RT,PCR results in the central slice showed negative histological findings. Six of these patients (24%) revealed positive RT,PCR results on the analysis of peripheral slices. However, three of these patients expressed only MelanA but not tyrosinase. Thirty lymph nodes from 24 nonmelanoma patients served as negative controls for RT,PCR. In three of these 24 patients (13%) expression of MelanA but not tyrosinase was detected by RT,PCR. Conclusions Molecular analysis of peripheral slices yielded six additional patients (10%) positive by RT,PCR who were initially negative by standard RT,PCR of the central slice. However, three of these six patients were found to express only MelanA but not tyrosinase. As MelanA expression was also found in 13% of control lymph nodes, positive MelanA expression alone in SLNs of melanoma patients requires cautious interpretation in order to avoid false-positive findings. Thus, additional molecular processing of peripheral slices did not significantly increase the number of patients with RT,PCR-positive SLNs. [source] Interleukin-4 and interleukin-13 enhance CCL26 production in a human keratinocyte cell line, HaCaT cellsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2005S. Kagami Summary Eotaxin-2/CCL24 and eotaxin-3/CCL26 are CC chemokines and their receptor, CC chemokine receptor 3 is preferentially expressed on eosinophils. It was reported that vascular endothelial cells and dermal fibroblasts produced CCL26. However, the regulation of CCL24 and CCL26 production in keratinocytes has not been well documented. We investigated the expression and production of CCL24 and CCL26 in the human keratinocyte cell line, HaCaT cells. Reverse transcription and polymerase chain reaction was performed using these cells and Enzyme-linked immunosorbent assay was carried out using supernatant of these cells. The production of CCL24 in HaCaT cells was slightly enhanced by IL-4 and that of CCL26 was strongly enhanced by IL-4 and IL-13. Furthermore, TNF-, generated a synergistic effect on IL-4 enhanced CCL26 production. Dexamethasone, IFN-, and the p38 mitogen-activated protein kinase inhibitor SB202190 inhibited IL-4 enhanced CCL26 production. IL-4 enhanced production of CCL26 was inhibited by leflunomide and JAK inhibitor 1, but not by JAK3 inhibitor, which indicates that it is mediated by JAK1-STAT6-dependent pathway. This result also strongly suggests the involvement of the type 2 IL-4 receptor in IL-4 enhanced production of CCL26. These results suggest that keratinocytes are involved in the migration of CC chemokine receptor 3 positive cells such as eosinophils in a Th2-dominant situation like atopic dermatitis. [source] Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cellsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2001S. Honda Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium. In this study, we examined the effects of IL-1,, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells. We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25,100 µg/ml) in the presence of IL-1, for 48 h. Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription,polymerase chain reaction (RT,PCR) methods were used to detect MT1-MMP mRNA. MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot. An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1,-stimulated synovial cells. In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1, treatment. KE-298 blocked this IL-1,-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1,-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL-1, results in the induction of MT1-MMP expression. Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE-298 may prevent this process by down-regulating MT1-MMP expression. [source] RELAXANT EFFECT OF ADRENOMEDULLIN ON BOVINE ISOLATED IRIS SPHINCTER MUSCLE UNDER RESTING CONDITIONSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2005Y Uchikawa SUMMARY 1.,The mechanisms involved in the fine adjustment of iris sphincter muscle tone are largely unknown. The aim of the present study was to clarify the effects of adrenomedullin on the resting tension of the bovine isolated iris sphincter muscle. 2.,The motor activity of the bovine isolated iris sphincter muscle was measured isometrically. The effects of adrenomedullin on resting tension were analysed in the presence of indomethacin. The presence of adrenomedullin mRNA in the preparation was determined by reverse transcription,polymerase chain reaction. Immunolabelling for adrenomedullin was also performed. 3.,Adrenomedullin significantly decreased the resting tension of the muscle. The relaxant effect of adrenomedullin was significantly inhibited by adrenomedullin (22,52), a putative antagonist for the adrenomedullin receptor, or calcitonin gene-related peptide (CGRP) (8,37), a putative antagonist for the CGRP1 receptor. The relaxant effect was almost completely blocked by a combination of adrenomedullin (22,52) and CGRP (8,37). 4.,The relaxant effect of adrenomedullin was also significantly diminished by 2,,5,-dideoxyadenosine, an inhibitor of adenylate cyclase, NG -nitro- l -arginine, an inhibitor of nitric oxide synthesis, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 5.,Reverse transcription,polymerase chain reaction analysis showed that adrenomedullin mRNA was expressed in the muscle strip. Immunopositive staining for adrenomedullin was detected in blood vessel cells and in the iris sphincter muscle cells. 6.,These results suggest that adrenomedullin may be an autocrine and paracrine regulator of the resting tension of the iris sphincter muscle. Its biological effects may be due to the direct involvement of adrenomedullin receptors and also to the stimulation of CGRP1 receptors. The stimulation of these receptors by the peptide leads to the activation of adenylate cyclase and soluble guanylate cyclase and subsequent relaxation of the muscle strip. [source] BRIEF EXPOSURE TO ETHANOL AUGMENTS VASCULAR CONTRACTILITY IN HUMAN CHORIONIC PLATE ARTERIESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2004Eun Hui Hong Summary 1.,Heavy alcohol consumption has been known as a risk factor for hypertension, although the mechanism by which alcohol intake causes hypertension remains elusive. 2.,We tested the hypothesis that brief exposure to ethanol augments vascular contractility through the stress response in human chorionic plate arteries. 3.,Human chorionic plate arteries were mounted in organ baths and exposed to 5% ethanol for 15, 30 or 45 min. 4.,Brief exposure for 45 min, but not 15 min, not only augmented contractility to KCl and 5-hydroxytryptamine 5 h after the end of exposure, but also increased the expression of heat shock protein (HSP) 70 in the tissues. 5.,Reverse transcription,polymerase chain reaction showed gradual increases of hsp70 mRNA expression, but not heat shock cognate 70 (hsc70), hsp90, or glucose regulatory protein 78 (grp78) mRNA expression, in an exposure time-dependent manner 3 h after the end of exposure. 6.,These results indicate that ethanol augments vascular contractility through the stress response. [source] Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell researchDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2010Tetsutaro Hayashi To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining "fluorescent activated cell sorting (FACS) index sorting" analysis and single-cell reverse transcription,polymerase chain reaction (RT,PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT,PCR. The results were plotted on the FACS sorting profile using the "index sorting" function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates. [source] Induction of initial heart ,-actin, smooth muscle ,-actin, in chick pregastrula epiblast: The role of hypoblast and fibroblast growth factor-8DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2008Hiroko Matsui During heart development at the gastrula stage, inhibition of bone morphogenetic protein (BMP) activity affects the heart specification but does not impair the expression of smooth muscle , -actin (SMA), which is first expressed in the heart mesoderm and recruited into initial heart myofibrils. Interaction of tissues between posterior epiblast and hypoblast at the early blastula stage is necessary to induce the expression of SMA, in which Nodal and Chordin are thought to be involved. Here we investigated the role of fibroblast growth factor-8 (FGF8) in the expression of SMA. In situ hybridization and reverse transcription,polymerase chain reaction showed that Fgf8b is expressed predominantly in the nascent hypoblast. Anti-FGF8b antibody inhibited the expression of SMA, cTNT, and Tbx5, which are BMP-independent heart mesoderm/early cardiomyocyte genes, but not Brachyury in cultured posterior blastoderm, and combined FGF8b and Nodal, but neither factor alone induced the expression of SMA in association with heart specific markers in cultured epiblast. Although FGF8b did not induce the upregulation of phospho-Smad2, anti-FGF8b properties suppressed phospho-Smad2 in cultured blastoderm. FGF8b was able to reverse the BMP-induced inhibition of cardiomyogenesis. The results suggest that FGF8b acts on the epiblast synergistically with Nodal at the pregastrula stage and may play a role in the expression of SMA during early cardiogenesis. [source] Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2006Noelia Andollo Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1-rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation-dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1-rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real-time reverse transcription,polymerase chain reaction analysis showed a higher expression of the U2af1-rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase-I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1-rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA-induced process of differentiation of ES cells. [source] Identification of asymmetrically localized transcripts along the animal,vegetal axis of the Xenopus eggDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2005Kensuke Kataoka In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40 000 independent embryonic cDNA clones, representing at least 17 000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription,polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns. [source] Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2000Akihiko Ishimura The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription,polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4 -injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo. [source] Spatio-temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: The presence of XVLG1 RNA in somatic cells as well as germline cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2000Kohji Ikenishi The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription,polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I,III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49,50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells. [source] Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3,-azido-3,-deoxythymidine and 2,,3,-dideoxyinosine,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2002Quanxin Meng Abstract Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3,-azido-2,,3,-dideoxythymidine (AZT) and 2,,3,-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667,12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 ,M or 300 ,M AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249,259; Meng Q et al. [2000b]: Toxicol Sci 54:322,329). Molecular analyses of HPRT mutant clones were performed by reverse transcription,mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 ,M AZT,ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency × percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 × 10,6 in HPRT and 6.0 × 10,6 in TK) at exposure levels of 100 ,M AZT,ddI (i.e., 1.94 × 10,6 in HPRT and 18.6 × 10,6 in TK) and 300 ,M AZT,ddI (i.e., 5.6 × 10,6 in HPRT and 34.6 × 10,6 in TK) (P < 0.05, Mann,Whitney U -statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 × 10,6) in cells exposed to 100 and 300 ,M AZT,ddI, respectively]. These results indicate that, at the same time that AZT,ddI potentiates therapeutic or prophylactic efficacy, the use of a second NRTI with AZT may confer a greater cancer risk, characterized by a spectrum of mutations that deviates from that produced solely by AZT. Environ. Mol. Mutagen. 39:282,295, 2002. Published 2002 Wiley-Liss, Inc. [source] Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+ENVIRONMENTAL MICROBIOLOGY, Issue 5 2001Roni Cohen The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1,P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+ -amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription,polymerase chain reaction (RT,PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+. [source] Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutantsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Jisung Ryu Abstract The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by ,-naphthoflavone (,NF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription,polymerase chain reaction. The concentration,dependent induction of CYP1A mRNA in the liver was observed after exposure to ,NF at nominal concentrations of 20, 100, and 500 ,g/ L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 ,g/L of ,NF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to ,NF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the ,NF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals. [source] | ||||||||||||||||||||||||||||||||||