Reverse Transcription-polymerase Chain Reaction Technique Research Articles

Identification of differentially expressed genes involved in spine formation on seeds ofDaucus carota L. (carrot), using annealing control primer (ACP) system

Carrot seeds normally have surface spines. The availability of a spineless mutant would be agronomically beneficial, eliminating the current efforts to remove those spines pre-sowing. Furthermore, the identification of spine-specific genes would provide insights into spine development in wild-type carrot seed. This effort could be facilitated through the use of an annealing control primer (ACP) system. Here, we employed a new and accurate reverse transcriptionpolymerase chain reaction (RT-PCR) that involves ACPs for identifying genes of interest. With these techniques, 11 expressed sequence tags (ESTs) were obtained for cloning and sequencing the genes that are differentially expressed in wild-type spiny seeds, but not in the spineless mutant. In all, 7 cDNAs exhibited significant sequence similarity with known genes from other species. These included cell wall-associated hydrolase, tail fiber assembly protein, transcriptional regulatory protein, berberine bridge enzyme, S-adenosyl methionine synthase, transketolase, and phenylalanyl t-RNA synthetase beta chain. Four other cDNA sequences had no significant identities with known genes. As revealed by RT-PCR, these genes regulate spine formation during the developmental stage. Our results suggest that PCR-based differential display RT-PCR techniques are a very useful tool for identifying spine-specific genes from carrot seeds.

Expression and Regulation of AT 1 Receptor in Rat Lung Microvascular Endothelial Cell

The renin-angiotensin system is thought to be involved in the development and progression of vascular endothelium inflammation, thereby contributing to vascular endothelium injury. To clarify the role of angiotensin II (Ang II) in rat pulmonary microvascular endothelial cells (RPMVECs), we examined the expression and functional significance of angiotensin II (Ang II) receptors in normal and lipopolysacchride (LPS) treated RPMVECs. The expressions of Ang II type 1(AT(1)) and Ang II type 2 (AT(2)) receptors in cultured RPMVECs were identified by the reverse transcription-polymerase chain reaction (RT-PCR) technique, Western blot and (125)I-labeled [Sar(1),Ile(8)] Ang II binding assays. The RPMVECs were treated with LPS (0.1-100 microg/ml) and Ang II (10(-8)-10(-5) M) for 24 h, respectively. Next, RPMVECs were treated with 10 microg/ml LPS or 10(-7) M Ang II for various times (3, 6, 12, and 24 h). The mRNA and protein levels of, AT(1) and AT(2) receptors, were evaluated at 3, 6, 12, and 24 h, respectively. The presence of specific Ang II binding sites in RPMVECs was found by Ang II saturated assays. RT-PCR revealed that only the AT(1) receptor mRNA is presented in RPMVECs. Western blot analysis of the RPMVECs protein extracts showed only one prominent band of the protein at approximately 41 KDa when probed with anti-AT(1) antibody and anti-AT(2) antibody. No AT(2) receptor mRNA and protein was detected. LPS treated cells resulted in an increase in the mRNA and protein levels of AT(1) receptor, whereas, Ang II treated cells showed a decrease in the mRNA and protein levels of AT(1) receptor. We found that primary cultured RPMVECs expressed only AT(1) receptor, but not AT(2) receptor. LPS up-regulated the transcriptional and post-transcriptional expression of AT(1) receptor in RPMVECS; in contrast, Ang II treatment caused a reduction in the mRNA and protein of AT(1) receptor in a time-dependent manner.

COMPARISON OF DRUG EFFLUX TRANSPORT KINETICS IN VARIOUS BLOOD-BRAIN BARRIER MODELS

The present study quantitatively compared the drug efflux transport kinetics of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent metabolite 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured bovine brain microvessel endothelial cells (BBMEC), and MDCK-MDR1 cells for 30 min in the presence or absence of the P-glycoprotein (P-gp) inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 microM). Multidrug resistance-associated protein (MRP) transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 microM), whereas the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared with the cultured cells. Because differences in the expression of P-gp, MRP5, and MRP6 were observed in the various BBB models using reverse transcription-polymerase chain reaction techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.

Simvastatin attenuates hypertrophic responses induced by cardiotrophin-1 via JAK–STAT pathway in cultured cardiomyocytes

Cardiotrophin-1 (CT-1) is a cytokine involved in the growth and survival of cardiac cells via activation of the Janus activated kinase/signal transducer activator of transcription (JAK/STAT). Statins, 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have effects that extend beyond cholesterol reduction and inhibit vascular smooth muscle cell (VSMC) proliferation and cardiac hypertrophy. However, whether stains also can inhibitin vitromyocardial hypertrophy or not still remains elusive. The purpose of this study was to explore the effects of simvastatin on the hypertrophy of cultured rat cardiomyocytes induced by CT-1 and to investigate whether this effect was mediated via JAK-STAT signaling pathway. Primary cardiomyocytes from 2-day-old (P2) rats were cultured, stimulated with CT-1, and treated with various concentration of simvastatin. Incorporation of [(3)H] leucine, reverse transcription-polymerase chain reaction and western blotting techniques were used to investigate cardiacmyocyte size, ANP mRNA and JAK-STAT protein expression. Simvastatin was proved, in a dose-independent manner, to decrease cardiacmyocytes size as well as protein synthesis, and inhibit ANP mRNA synthesis and JAK-STAT protein expression induced by CT-1 in cardiacmyocytes. These results suggest that simvastatin can ameliorate cardiacmyocytes hypertrophyin vitrovia JAK-STAT signaling pathways. The present study provides a novel understanding and alternative therapeutic strategy for cardiac hypertrophy.

Thrombin-like enzymes from venom gland of Deinagkistrodon acutus: cDNA cloning, mechanism of diversity and phylogenetic tree construction1

To clone cDNAs of thrombin-like enzymes (TLEs) from venom gland of Deinagkistrodon acutus and analyze the mechanisms by which their structural diversity arose. Reverse transcription-polymerase chain reaction and gene cloning techniques were used, and the cloned sequences were analyzed by using bioinformatics tools. Novel cDNAs of snake venom TLEs were cloned. The possibilities of post-transcriptional recombination and horizontal gene transfer are discussed. A phylogenetic tree was constructed. The cDNAs of snake venom TLEs exhibit great diversification. There are several types of structural variations. These variations may be attributable to certain mechanisms including recombination.

Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control.

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

Clinical significance of MUC1 and c-Met RT-PCR detection of circulating tumor cells in patients with gastric carcinoma

Background Recently, several reverse transcription-polymerase chain reaction (RT-PCR) techniques have been proven to be useful in the detection of circulating tumor cells (CTCs) in cancer patients. We attempted to detect CTCs in patients with gastric cancer (GC) using a RT-PCR assay for c-Met and MUC1 and to evaluate their clinical value. Methods Using a RT-PCR assay, c-Met and MUC1 mRNAs were amplified in 52 GC patients and 36 healthy individuals. Analyses were carried out for their correlation with the patients' clinicopathologic features, the occurrence of new post-operative metastasis, as well as the overall survival rates. Results In the RT-PCR analysis of peripheral blood, 61.5% (32 / 52) and 71.2% (37 / 52) of GC patients were positive for c-Met and MUC1 mRNA, respectively. The sensitivity and specificity of either mRNA detected in peripheral blood is 82.7% and 86.1%, respectively, with an accuracy of 84.1%. The detection of c-Met or MUC1 mRNA was significantly correlated with the depth of tumor invasion, lymph node metastases, TNM stage, vessel invasion, perineural involvement, and post-operative metastasis (all P < 0.05). Kaplan–Meier analysis demonstrated that the overall survival rate of patients with positive c-Met or MUC1 mRNA expression in the peripheral blood was significantly shorter than in patients negative for c-Met or MUC1 mRNA expression (both P < 0.05). Conclusions Our findings suggest that using RT-PCR for the detection of c-Met or MUC1 mRNA may be a promising tool for the early detection of micro-metastatic CTCs in GC patients. Combination of these 2 tumor-specific mRNA markers would increase the detection rate and may be clinically helpful in predicting the outcome in GC patients.

Expression and Localization of Fas Ligand and Fas During Atresia in Porcine Ovarian Follicles

To reveal the mechanisms regulating the selective atresia of follicles in porcine ovaries, we examined the changes in the mRNA and protein levels of cell-death ligand, Fas/APO-1/CD95 ligand (FasL), and its receptor, Fas/APO-1/CD95 (Fas), and the localization of the proteins in granulosa cells during follicular atresia using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, respectively. Trace levels of FasL mRNA and protein were detected in the granulosa cells of healthy follicles; however, weak levels were detected in those of early atretic follicles, and the levels increased during atresia. Trace/weak levels of Fas mRNA and protein were detected in the granulosa cells of healthy follicles. Fas protein was located in the cytoplasmic area, not in cell membrane area, indicating that it has no activity in regard to inducing apoptosis. When apoptosis commences in granulosa cells, Fas moves from the cytoplasmic to cell membrane area. FasL and Fas mRNAs and proteins in granulosa cells were upregulated during follicular atresia. The FasL and Fas system may play a crucial role in the regulation of apoptosis in granulosa cells during selective follicular atresia in porcine ovaries.

Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas

The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1%) were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

Plasmodium chabaudi: Reverse transcription PCR for the detection and quantification of transmission stage malaria parasites

We have developed two reverse transcription polymerase chain reaction (RT-PCR) techniques to detect and quantify the transmission stages (gametocytes) of Plasmodium chabaudi malaria parasites. Both the qualitative and quantitative techniques are based on the amplification of mRNA coding for the P. chabaudi protein Pcs230, which is expressed exclusively in gametocytes. The quantitative RT-PCR (qRT-PCR) technique was developed and validated by examining serial dilutions of known gametocyte densities. The method generated a high correlation between calibration curves of blind samples ( R 2 = 0.86). The technique was found to be specific, reproducible, and time efficient for quantification of both patent and sub-patent gametocytemia with a sensitivity level 100–1000 times greater than microscopy. The qualitative RT-PCR (RT-PCR) technique was used to monitor the persistence and dynamics of P. chabaudi gametocytes following acute infection. Mice in two independent experiments were sampled for up to 87 days post-infection. RT-PCR showed that gametocytes can persist for up to 8 weeks, post-infection, whereas microscopy could only detect gametocytes up to 6 weeks. Potential applications of the above techniques for studying the ecology, evolution, and epidemiology of malaria transmission are discussed.

Cooling‐induced contraction of the rat gastric fundus: Mediation via transient receptor potential (TRP) cation channel TRPM8 receptor and Rho‐kinase activation

1. Cooling has been shown to induce contractions of several smooth muscles in vitro. However, the mechanism involved in the response is not yet known. In the present study, we investigated the possible involvement of transient receptor potential (TRP) cation channel TRPM8 receptors and the Rho-kinase pathway in cooling-induced contraction of the rat fundus. 2. Cooling-induced contractions were inversely proportional to temperature. Contractions were significantly reduced (by 65.6 +/- 2.4%; P < 0.05) in a Ca2+-free (1 mmol/L EGTA) medium, but were not significantly inhibited by nifedipine (10(-6) mol/L). 3. Capsazepine (3 x 10(-6) and 3 x 10(-5) mol/L), a TRPM8 receptor antagonist, inhibited cooling-induced contraction of the rat gastric fundus. 4. The Rho-kinase inhibitor Y-27632 concentration-dependently inhibited cooling-induced contraction of the gastric fundus, producing approximately 90% inhibition at a concentration of 10(-5) mol/L. Contractions were also inhibited by genistein (3 x 10(-5) mol/L), a tyrosine kinase inhibitor, but not by GF 109203X (10(-7) mol/L), a protein kinase C inhibitor. 5. Using reverse transcription-polymerase chain reaction techniques, it was observed that the mRNA for the TRPM8 receptor and Rho-kinase were expressed in the rat gastric fundus. 6. These results would suggest that cooling-induced contraction of the rat fundus is mediated by activation of TRPM8 receptors via a mechanism involving activation of Rho-kinase.

Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS

As new assay methods for quantitative reverse transcription-polymerase chain reaction (RT-PCR), such as real time RT-PCR techniques, approach theoretical limits of per reaction sensitivity, further increments in the sensitivity of measurements of viral load can only be achieved by increasing the amount of input RNA per reaction. We describe a robust, convenient, rapid integrated approach for specimen preparation and real time RT-PCR assay for plasma simian immunodeficiency virus (SIV) RNA viral load that provides a threshold sensitivity of 10 copy Eq/ml, and tolerates less than optimally processed specimens. The method provides accurate quantitation of viral load for the SIV virus isolates in common use for non-human primate studies. We demonstrate the utility of the method in sensitively tracking viral load in an animal showing effective control of viral replication to levels below the threshold for quantitation in conventional assays.

Methylation‐Associated Silencing of Death‐Associated Protein Kinase Gene in Laryngeal Squamous Cell Cancer

Death-associated protein kinase (DAPK) is a Ca/calmodulin-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It has been found that DAPK gene is frequently inactivated by its promoter hypermethylation in some cancers and tumor cell lines. However, it is not clear whether promoter hypermethylation of DAPK gene exists in laryngeal squamous cell cancer (LSCC). The aim of this study was to investigate the promoter methylation status of the DAPK gene in LSCC and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on Hep-2 cells, a human laryngeal cancer cell line, and on xenografts of Hep-2. Methylation-specific polymerase chain reaction (PCR) and reverse-transcription PCR techniques were used to determine the promoter methylation status and mRNA expression of DAPK gene in LSCC. Furthermore, Hep-2 cells in vitro and in vivo were treated by 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth. Hypermethylation of DAPK gene promoter was found in 39 (67.2%) of 58 LSCC samples. There was no significant difference in the promoter hypermethylation rate among the samples of different histologic grades or samples from patients with different T stages. However, there was significant difference in methylation status of DAPK gene between the samples from patients in N0 stages and those from patients in N1 stages. No promoter hypermethylation of DAPK gene was found in any of the five normal laryngeal tissue samples. DAPK mRNA expression was not detected in tumor specimens with promoter hypermethylation. On the contrary, DAPK mRNA expression was observed in the unmethylated tumor specimens, specimens from tissues adjacent to the tumor, and normal laryngeal tissues samples. Promoter hypermethylation of DAPK gene was found, and no DAPK mRNA expression was detected in Hep-2 cells. DAPK mRNA expression in Hep-2 cells and xenografts could be restored by treating cells and xenografts with 5-Aza-CdR. The tumors' xenografts, induced by way of Hep-2 cell injection in nude mice treated with 5-Aza-CdR, were obviously smaller than those in nude mice treated with phosphate-buffered saline. Abnormal loss of DAPK expression could be associated with aberrant promoter region methylation in the LSCC. 5-Aza-CdR may slow the growth of Hep-2 cells in vitro and in vivo by reactivating tumor suppressor gene DAPK silenced by de novo methylation.

Detection of Human Coronavirus-NL63 in Children in Japan

Human coronavirus NL63 recently found in The Netherlands has been detected in Japan with a reverse transcription-polymerase chain reaction technique in clinical specimens from pediatric patients with respiratory symptoms. Of 419 specimens that were negative for common respiratory viruses, 5 were positive for human coronavirus NL63, and these specimens were all collected in the first 3 months of 2003.

Pregnancy-induced decrease in the relaxant effect of terbutaline in the late-pregnant rat myometrium: role of G-protein activation and progesterone

The effectiveness of beta2-agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the beta-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the beta-adrenergic receptors was investigated by a radiolabelled GTP binding assay.EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in beta-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the beta2-adrenergic receptors and their mRNA level and increased the G-protein-activating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after beta2-agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of beta2-adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone.

First Report on the Natural Occurrence of Cherry virus A in Mirabelle Plum (Prunus domestica var. insititia).

Cherry virus A (CVA) is a member of the Capillovirus genus (2). It was discovered serendipitously during cloning of the little cherry agent (2) and has since been shown to be relatively widespread in sweet and sour cherry (Prunus cerasus and P. avium) (2,3). It is currently unclear whether CVA is associated with any specific symptoms in these hosts. Although it can be transmitted by grafting and thus propagated in peach, it has not been reported to naturally infect any host other than cherry. Using a degenerate reverse transcription-polymerase chain reaction (RT-PCR) technique targeting a conserved region of the RNA-dependent RNA polymerase (RdRp) and allowing the amplification of members of the Trichovirus, Capillovirus, and Foveavirus genera of filamentous plant viruses (1), a number of symptomatic Prunus spp. germplasm were evaluated. Among these, a cv. Mirabelle dorée accession (Prunus domestica var. insititia P332) of French origin exhibited severe symptoms of rosetting, severe leaf and fruit deformation, and yellow mosaic occasionally turning necrotic. RT-PCR conducted on symptomatic samples produced an amplification product of the expected size (362 bp) in several independent experiments. Sequencing of these products yielded a single sequence (GenBank Accession No. AY792509) with 88.1% nucleotide identity and 93.2% amino acid identity with the type strain of CVA (2). Presence of a CVA isolate was independently confirmed using a CVA-specific PCR assay directly on the original plum material or following experimental transmission by grafting on several new hosts including apricot (P. armeniaca cv. Priana) and plum (P. domestica cv. Prune d'Ente). To our knowledge, this is the first report of natural infection of CVA in plum. The symptoms observed in the infected plum are reminiscent of those caused by severe Prune dwarf virus (PDV) strains. Infection by PDV was confirmed using a PDV-specific PCR assay. The contribution, if any, of CVA to the symptoms observed remains to be evaluated. These findings suggest that the possible presence of CVA in noncherry Prunus spp. hosts should be taken into consideration by quarantine and certification programs.

Identification of a novel zebrafish SULT1 cytosolic sulfotransferase: Cloning, expression, characterization, and developmental expression study

By searching the zebrafish expressed sequence tag database, we had identified two partial cDNA clones encoding the 5′- and 3′-regions of a putative cytosolic sulfotransferase (SULT). Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, a full-length cDNA encoding this zebrafish SULT was amplified, cloned, and sequenced. Analysis of the sequence data revealed that this novel zebrafish SULT displays 49, 46, and 45% amino acid sequence identity to human SULT1A1, mouse SULT1D1, and rat SULT1C1. This zebrafish SULT therefore appears to belong to the SULT1 cytosolic SULT gene family. Recombinant zebrafish SULT (designated SULT1 isoform 4), expressed using the pGEX-2TK prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 35 kDa protein upon sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Among the endogenous compounds tested as substrates, the purified SULT1 isoform 4 displayed significant sulfating activities toward thyroid hormones, estrone, and dehydroepiandrosterone. The enzyme also showed activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds, with a pH optimum at 7.0. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 28 and 37 °C. Among 10 divalent metal cations tested, Fe 2+, Hg 2+, Co 2+, Zn 2+, Cu 2+, and Cd 2+ exhibited dramatic inhibitory effects on the activity of the enzyme. Developmental expression study using RT-PCR revealed that the zebrafish SULT1 isoform 4 showed a low level of expression in the segmentation period during the embryonic development, which gradually increased to a high level of expression throughout the larval stage onto maturity.

Short-term effects of endocrine-disrupting chemicals on the expression of estrogen-responsive genes in male medaka ( Oryzias latipes)

To evaluate the estrogenic activities of selected estrogenic compounds such as estradiol-17β (E2), nonylphenol (NP), 4-(1-adamantyl)phenol (AdP), bisphenol A (BPA), BPA metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) and 4,4′-dihydroxy-α-methylstilbene (DHMS) in the shortest possible time, we investigated the expression of estrogen-responsive genes such as vitellogenin I, vitellogenin II and α-type estrogen receptor genes in the liver of male medaka ( Oryzias latipes) using reverse transcription-polymerase chain reaction (RT-PCR) techniques. These estrogen-responsive genes responded rapidly to selected estrogenic compounds after 8 h exposure, and the expression of hepatic vitellogenin II and estrogen receptor α mRNA was found to be more responsive than that of vitellogenin I mRNA. As a result, the relative estrogenic potencies of tested chemicals descended in the order of E2 (100) > MBP (0.38) > AdP (0.25) > DHMS (0.05) > NP (0.02) > BPA (0.001). Moreover, this preliminary study indicates that AdP and DHMS should be considered as candidate estrogenic compounds with the potential to induce hepatic estrogen-responsive genes in male medaka. These results suggest that vitellogenin I, vitellogenin II and estrogen receptor α gene expression patterns alter in male medaka treated with selected estrogenic compounds, and that these genes may be useful molecular biomarkers for screening estrogenic endocrine-disrupting chemicals in the shortest possible time.

Effect of Selenium on Expression of Selenoproteins in Mouse Fibrosarcoma Cells

Selenium (Se), an essential trace element, is incorporated into selenoproteins as selenocysteine using insertion machinery, including UGA codon and selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region (3'-UTR) of mRNA. To assess the biological effects of tumor cells exposed to the elevated, but nontoxic Se level on glutathione peroxidase (GPx1 [cellular] and GPx3 [extracellular]), thioredoxin reductase (TrxR), and selenoprotein P (SeP) mRNA expression, we introduced a semiquantitative reverse transcription-polymerase chain reaction technique for each selenoprotein transcript using beta-actin as a reference housekeeping gene in mouse fibroblasts (WEHI 164). Cell lines were cultured with 1.0, 2.5, and 5.0 ng of Se in 1 mL of medium for 3 and 7 d, apart from the control cell line with standard medium. It was found that Se exerts a statistically significant (p<0.05) effect only on GPx3 mRNA, referred to as the optical density (OD) ratio (GPx3/beta-actin). Moreover, the lowest Se level affected GPx3 mRNA expression more strongly than its highest concentrations. In an in vitro model applied in this study, GPx3 gene expression is most specific for Se supplementation.

Detection of FUS-CHOP fusion gene in paraffin-embedded tissues and its clinicopathologic significance for myxoid/round cell liposarcomas

To explore the feasibility of detecting FUS-CHOP fusion gene in formalin-fixed, paraffin-embedded tissue and its application in the diagnosis and differential diagnosis of myxoid/round cell liposarcomas (MRCLs). Forty-four formalin-fixed, paraffin-embedded MRCL samples and 60 control cases (atypical/well-differentiated liposarcoma, pleomorphic liposarcoma, low-grade myofibrosarcoma, etc.) retrieved from the archival files were studied. Nested reverse transcription-polymerase chain reaction (RT-PCR) technique was employed to detect the FUS-CHOP mRNA expression, followed by DNA sequencing confirmation of the PCR product. Housekeeping gene PGK was used to assess the quality of the mRNA templates. PGK mRNA was detected in 93 of 104 tumor cases (89.4%), including 39 MRCLs cases (39/44, 88.6%) and 90% of the negative control cases. Type II FUS-CHOP fusion transcript was successfully detected in 20 out of 39 (51.3%) MRCL cases. Type I FUS-CHOP fusion transcript was not detected in any MRCLs in this study. All 60 negative control cases were negative for the FUS-CHOP fusion gene transcripts. (1) Nested RT-PCR can be used to detect FUS-CHOP mRNA in formalin-fixed, paraffin-embedded tissues. (2) FUS-CHOP is considered a specific molecular and genetic hallmark for MRCLs. Nested RT-PCR is a sensitive and specific technique in detecting FUS-CHOP gene, and can be used in the diagnosis and differential diagnosis of MRCLs.