Competitive RT-PCR Research Articles

Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease

BackgroundDupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures.ResultsThe Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis.ConclusionThese data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of β-catenin expression in DD.

Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood

Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.

A system for receptor functional analysis based on c-fos mRNA expression: Analysis of GnRH receptors as a test system

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.

A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA

There are several methods available to analyze the mRNA concentration quantitatively. Among them, the competitive reverse transcription (RT-)PCR method is very useful. For this method, Cy5-labeled primers were used, and after gel electrophoresis in 7 M urea, the Cy5-labeled single-strand DNA was measured by a fluorescence detector. However, as the equipment to measure the Cy5-labeled fluorescence is expensive, we developed a new method using SYBR Gold staining. After gel electrophoresis in 7 M urea, the single-strand PCR product DNA was stained with SYBR Gold, and photographed with a standard UV-transilluminator and a standard digital camera with a specific filter for SYBR Gold staining. The photographic image was digitized by an imaging software. We measured beta-actin and plasma glutathione peroxidase (Gpx3) mRNA concentrations of HepG2 cell cultured at 5 and 20% oxygen tension. The Gpx3 expression was increased by hypoxia. The result was equivalent to the data obtained by the real-time PCR analysis.

The use of genetic programming in the analysis of quantitative gene expression profiles for identification of nodal status in bladder cancer

BackgroundPrevious studies on bladder cancer have shown nodal involvement to be an independent indicator of prognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis from molecular profiles of primary urothelial carcinoma tissues.MethodsThe study included primary bladder tumor tissues from 60 patients across different stages and 5 control tissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positive and node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardized competitive RT-PCR and the expression values of the training set samples were run through an iterative machine learning process called genetic programming that employed an N-fold cross validation technique to generate classifier rules of limited complexity. These were then used in a voting algorithm to classify the validation set samples into those associated with or without nodal metastasis.ResultsThe generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when compared to the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in gene expression motifs that cumulatively suggested a pattern ICAM1>MAP2K6>KDR for node positive cases. Additionally, the motifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors. Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producing an accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis.ConclusionOur study demonstrates the use of standardized quantitative gene expression values from primary bladder tumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status. Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematical combinations in the progression towards nodal positivity. Further studies are needed to identify more class-specific signatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

Growth Hormone-Releasing Hormone (GHRH) Neurons in the Arcuate Nucleus (Arc) of the Hypothalamus Are Decreased in Transgenic Rats Whose Expression of Ghrelin Receptor Is Attenuated: Evidence that Ghrelin Receptor Is Involved in the Up-Regulation of GHRH Expression in the Arc

GH secretagogue (GHS)/ghrelin stimulates GH secretion by binding mainly to its receptor (GHS-R) on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus. GHRH, somatostatin, and neuropeptide Y (NPY) in the hypothalamus are involved in the regulatory mechanism of GH secretion. We previously created transgenic (Tg) rats whose GHS-R expression is reduced in the Arc, showing lower body weight and shorter nose-tail length. GH secretion is decreased in female Tg rats. To clarify how GHS-R affects GHRH expression in the Arc, we compared the numbers of GHS-R-positive, GHRH, and NPY neurons between Tg and wild-type rats. Immunohistochemical analysis showed that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were reduced in Tg rats, whereas the numbers of NPY neurons and GHS-R-positive NPY neurons did not differ between the two groups. The numbers of Fos-positive neurons and Fos-positive GHRH neurons in response to KP-102 were decreased in Tg rats. Competitive RT-PCR analysis of GHRH mRNA expression in the cultured hypothalamic neurons showed that KP-102 increased NPY mRNA expression level and that NPY decreased GHRH mRNA expression level. KP-102 increased GHRH mRNA expression level in the presence of anti-NPY IgG. GH increased somatostatin mRNA expression. Furthermore, GH and somatostatin decreased GHRH mRNA expression, whereas KP-102 showed no significant effect on somatostatin mRNA expression. These results suggest that GHS-R is involved in the up-regulation of GHRH and NPY expression and that NPY, somatostatin, and GH suppress GHRH expression. It is also suggested that the reduction of GHRH neurons of Tg rats is induced by a decrease in GHS-R expression.

Design, development and evaluation of a competitive RT-PCR for quantitation of GBV-C RNA

Although GB virus C (GBV-C) hepatocyte pathogenicity is still controversial, it appears that at least some strains of this virus are lymphotropic. During the past few years, several reports have documented an apparently beneficial role played by GBV-C in the course of HIV-1 infection. At present, a commercial kit for GBV-C RNA quantitation is not available. In this study, a competitive RT-PCR method for GBV-C in serum samples is described. The sensitivity of the assay proved to be 10 4 and 10 3 genomic equivalents for positive and negative sense RNAs, respectively. This method will discriminate specifically between positive and negative strand RNAs with a discrimination index of at least five log 10. Out of 60 samples from different hematological disorders ( n = 49), HIV-1 positive patients ( n = 7), and blood donors ( n = 4), 10 proved to be GBV-C RNA positive. Viral load ranged from 1.1 × 10 7 to 2.34 × 10 8 genomic equivalents/ml. Such values correlated linearly ( r = 0.986) with those obtained by a 10-fold serial dilution method. In studies exploring the GBV-C pathogenicity, the measurement of viral load may contribute to understand the possible mechanisms involved.

Role of Angiotensin II and Endothelin‐1 Receptors in Aging‐Related Functional Changes in Rat Cardiovascular System

Angiotensin II (AII) and endothelin-1 (ET-1) are regarded as key players in the age-related changes in cardiovascular function. They are known to be involved in the pathogenesis of cardiac fibrosis and coronary vascular atherosclerosis. AII- and ET-induced vasoconstriction was augmented in coronary arteries of Langendorff-perfused heart from aged rats. In papillary muscles, ET-1-induced positive inotropic effect (PIE) was diminished by aging. On the other hand, both ET-1 and AII caused greater vasoconstriction in aged rat coronary arteries compared to those in the young rat. To further elucidate the mechanism of these age-dependent changes in cardiovascular effects of ET-1 and AII, we examined the expression of AII and ET-1 receptors in young (2-month-old) and aged (24-month-old) rats. Total RNA was isolated from left ventricles. For determination of the gene expression of AT(1) receptor and ET(A)/ET(B) receptor mRNA, competitive RT-PCR and Northern blot analysis were performed, respectively. [(125)I]ET-1 receptor assay was carried out in left ventricular membrane fraction. AT(1)-receptor, ET(A)-, and ET(B)-receptor mRNA were upregulated in the left ventricles of senescent rats compared with young ones. The affinity of ET-1-receptor was not changed, but receptor density was significantly increased in aged rats. Although the precise mechanism for the upregulation of AT(1) receptor and ET-1 receptor in the aged rat heart has not been clarified yet, these findings suggest that the activation of the renin-angiotensin system as well as ET receptor may be important for the physiological changes in aged hearts.

Chondrocyte gene expression in osteoarthritis: Correlation with disease severity

Osteoarthritis is characterized by a progressive degradation of cartilage structure and function. This study tests the hypothesis that disease severity is characterized by alterations in expression of cartilage-specific genes for aggrecan and collagen type II. Cartilage, discarded from six subjects undergoing knee replacement, was subdivided into homogeneous portions by the surgeon according to the Outerbridge classification. For four subjects, it was possible to separate the tissue into two or three fractions with different disease severity. Portions of each sample were prepared either for histological analysis and ranking according to the Mankin system or for RNA extraction. Quantitative, competitive RT-PCR assays were used for measurement of mRNA for aggrecan, collagen type II, and glyceraldehyde-3-phosphate dehydrogenase. Clinical grading was correlated with histological score (Spearman r = 0.60, p = 0.043). There was a striking decrease in expression of aggrecan and collagen II that was correlated with increase in the grade in regions of cartilage within an individual subject. In the series of 12 samples, there was an inverse correlation between aggrecan expression and osteoarthritis grade (Spearman r = -0.59, p = 0.042). In conclusion, there was an inverse relationship between regional disease severity in osteoarthritis and expression of aggrecan. Use of quantitative, competitive RT-PCR is practical for assessment of chondrocyte gene signatures.

Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells

Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-kappaB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response.

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0–210 μM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 μM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0–210 μM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.

Vascular Endothelial Growth Factor Antagonist Modulates Leukocyte Trafficking and Protects Mouse Livers against Ischemia/Reperfusion Injury

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.

Effects of oxidized-low density lipoprotein on expression of urotensin II receptor GPR14 in rat aortic smooth muscle cells

To investigate the effects of oxidized-low density lipoprotein (ox-LDL) and urotensin II (U II) on the proliferation of rat aortic smooth muscle cells (VSMCs) and the effect of ox-LDL on the mRNA expression of U II receptor GPR14 in. Rat VSMCs were incubated with UII and ox-LDL at different concentrations. The viability of rat VSMCs was detected by MTT assay. Rat VSMCs were incubated with ox-LDL at the concentrations of 0, 0.1, 1, 10, and 50 microg/ml. Twelve hours later competitive RT-PCR was used to detect the effects of concentration of ox-LDP on the mRNA expression of GPR14 in the VSMCs. Another VSMCs were incubated with ox-LDL at the final concentration of 50 microg/ml, and 0, 2, 6, 12, and 24 hours later competitive RT-PCR was used to detect the mRNA expression of GPR14 in the VSMCs. (125)I-labelled U II was added into the culture fluid of VSMCs, and radioligand binding assay was used to calculate the maximum binding (B(max)). UII of the concentration of 50 nmol/L significantly increased the proliferation of VSMCs. When the concentration of ox-LDL was 10 microg/ml the proliferation of VSMCs (P < 0.01) was 0.678 +/- 0.061, increased by 121% compared with the control group (0.325 +/- 0.052, P < 0.01). 0.01 microg/ml of ox-LDL and 10 nmol/L of UII showed a synergistic effect on the proliferation of VSMCs with the MTT value increased to 162% +/- 29% that of the control group (P < 0.01). When the concentration of ox-LDL increase to 0.1 microg/ml the synergistic effect was attenuated with the MTT value of 153% +/- 22% that of the control group (P < 0.05) and when the concentration of ox-LDL increased to 1 microg/ml the synergistic effect diminished, with a MTT value of 123% +/- 13% that of the control group (P > 0.05). When the concentration of ox-LDL was 50 microg/ml the proliferation of VSMCs the MTT value decreased to 59% that of the of control group, however, ox-LDL of the concentration of 50 microg/ml combined with UII of the concentration of 10 nmol/L the MTT value increased to 68% that of the control group, showing that UII of that concentration significantly inhibited the cytotoxic effect of ox-LDL. Incubation of VSMCs with the ox-LDL at the concentration of 0.1 microg/ml the GPR14 mRNA expression of the VSMCs was 125.1% that of the control group (P < 0.01) as assessed by competitive RT-PCR, however, when the concentration of ox-LDL increased over 1 microg/ml the GPR14 mRNA expression does-dependently decreased as much as 72.6% that of the control group (all P < 0.01), with the maximal effect when the concentration of ox-LDL reached 50 microg/ml. ox-LDL at the concentration of 50 microg/ml time-dependently down-regulated the GPR14 mRNA expression with the maximal effect 12 hours after addition of ox-LDL and this effect was sustained for up to 24 hours. After incubation for 12 hours the binding of (125)I-UII to VSMCs was 117.5% that of the control group (P < 0.01) with the ox-LDL at the concentration of 0.1 microg/ml, and was 68.8% that of the control group (P < 0.01) with the ox-LDL at the concentration of 50 microg/ml. ox-LDL and with U-II. GPR14 at certain concentrations show synergetic effects on the proliferation of VSMCs. The GPR14 mRNA expression in VSMCs can be upregulated by low concentration of ox-LDL, while downregulated by high concentration of ox-LDL.

Competitive RT-PCR: Preparation of Competitor RNA

INTRODUCTION The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor. This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest. Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out, as described in Comptetitive RT-PCR: Estimation of Copy Number.

The use of moderate hypothermia during cardiac surgery is associated with repression of tumour necrosis factor-α via inhibition of activating protein-1: an experimental study

IntroductionThe use of moderate hypothermia during experimental cardiac surgery is associated with decreased expression of tumour necrosis factor (TNF)-α in myocardium and with myocardial protection. In order to identify the cellular mechanisms that lead to that repression, we investigated the effect of hypothermia during cardiac surgery on both main signalling pathways involved in systemic inflammation, namely the nuclear factor-κB (NF-κB) and activating protein-1 pathways.MethodTwelve female pigs were randomly subjected to standardized cardiopulmonary bypass with moderate hypothermia or normothermia (temperature 28°C and 37°C, respectively; six pigs in each group). Myocardial probes were sampled from the right ventricle before, during and 6 hours after bypass. We detected mRNA encoding TNF-α by competitive RT-PCR and measured protein levels of TNF-α, inducible nitric oxide synthase and cyclo-oxygenase-2 by Western blotting. Finally, we assessed the activation of NF-κB and activating protein-1, as well as phosphorylation of p38 mitogen-activated protein kinase by electrophoretic mobility shift assay with super shift and/or Western blot.ResultsDuring and after cardiac surgery, animals subjected to hypothermia exhibited lower expression of TNF-α and cyclo-oxygenase-2 but not of inducible nitric oxide synthase. This was associated with lower activation of p38 mitogen-activated protein kinase and of its downstream effector activating protein-1 in hypothermic animals. In contrast, NF-κB activity was no different between groups.ConclusionThese findings indicate that the repression of TNF-α associated with moderate hypothermia during cardiac surgery is associated with inhibition of the mitogen-activated protein kinase p38/activating protein-1 pathway and not with inhibition of NF-κB. The use of moderate hypothermia during cardiac surgery may mitigate the perioperative systemic inflammatory response and its complications.

High and persistent excretion of hepatitis A virus in immunocompetent patients

The duration and level of virus excretion in blood and faeces of patients with hepatitis A virus (HAV) infection were studied in relation to levels of alanine aminotransferase (ALT), disease severity and HAV genotype. Clinical data, blood and faeces were collected from 27 patients with acute hepatitis A (median age: 33 years) for a maximum of 26 weeks. Single blood donations from 55 other patients with acute HAV (median age: 32 years) were also used. Virus loads were quantified by competitive nested RT-PCR. HAV was excreted in faeces for a median period of 81 days after disease onset, with 50% of patients still excreting high levels at Day 36 (2 x 10(6) - 2 x 10(8) copies/ml faeces suspension). Viraemia was detected, but not quantifiable, for a median period of 42 days. In the first 10 days of illness, higher ALT levels were correlated with higher viraemia levels. Comparison of patients infected with genotype 1a with those infected with type 1b did not differ significantly in terms of the duration of HAV excretion or jaundice. In conclusion, faecal excretion of HAV is at a high titre in the first month, perhaps making patients infectious for a longer period than assumed currently. Blood banks should be aware that viraemia may be present for more than 1 month, and genotype did not affect the duration of virus excretion or jaundice.

The Analysis of the Autoinducer Gene Expression Related Quorum Sensing Mechanism in Catheter Associated Urinary Tract Infection

Purpose: Catheter associated urinary tract infection (CAUTI) frequently occurs in the patients with an indwelling Foley catheter, and it can cause serious morbidity or mortality. However, there have been no reports about quorum sensing mechanisms in indwelling Foley catheter. It's our purpose to find out the quorum sensing mechanisms of isolated bacteria from biofilm in Foley catheters. Materials and Methods: Silicone Foley catheters were placed in 90 patients with neurogenic bladders. At the 3rd, 5th, 7th, 14th and 30th day after the catheters were placed, the catheters were removed and the biofilm formations were evaluated by routine culture and microscopy. The ygaG gene, which was reported to be an autoinducer synthase gene was carried out cloning in E. coli. The quantity of the mRNA expression of the ygaG gene was analyzed according to the time by competitive reverse transcription polymerase chain reaction (RT-PCR). Results: 289 different types of bacteria were isolated by cultivation. The most common species were Pseudomonas, Klebsiella, Serratia, Proteus and Escherichia species. The autoinducer synthase gene, such as the ygaG gene for Escherichia coli, was detected by RT-PCR. On competitive RT-PCR of the ygaG gene, the mRNA expression was 3.77x10copies/μl at the 3rd day, 5.94x10copies/μl at the 5th day, 8.07x10copies/μl at the 7th day, 2.51x10copies/μl at the 14th day and 6.81x10copies/μl at the 30th day. Therefore, the expression of the autoinducer synthase gene was observed at the early insertion period and it was then maintained. Conclusions: This is the first study to document the autoinducer synthase gene expression associated quorum sensing mechanism in CAUTI. The quorum sensing mechanism may be a new target for the management of CAUTI. (Korean J Urol 2006;47:945-952)

Effects of adenovirus-mediated gene transfer of ICOSIg and CTLA4Ig fusion protein on experimental autoimmune myocarditis

Objective: To explore the therapeutic alliance effects of adenovirus vector-mediated gene transfer of ICOSIg and CTLA4Ig fusion protein on experimental autoimmune myocarditis (EAM).Methods: Expression vector pAdeno-CTLA4Ig and pAdeno-ICOSIg was constructed and transfected into HEK293 cells. Adenovirus expresses CTLA4Ig and ICOSIg was produced. Ad-CMV-GFP was used as controls. EAM was induced in Lewis rats by injection of procine cardiac myosin. All the immunized rats were divided into four groups. Group A (n = 15) received adenovirus containing CTLA4Ig and ICOSIg from day 14–28; group B (n = 15), group C (n = 15) and group D (n = 15) received adenovirus containing CTLA4Ig, ICOSIg and GFP, respectively. Group E (n = 10) was normal controls never received immunization. On day 28, all the rats were killed after echocardiography examination. Histopathological examination was used to observe inflammation in the myocardium. Western blot was used to detect CTLA4, ICOS, ICOSL and competitive RT-PCR for B7-1, B7-2 expression. T lymphocyte proliferation assay was performed and ELISPOT was used to detect the Th1 and Th2 production.Results: Alliance application of CTLA4Ig and ICOSIg exerts therapeutic effects on EAM. After a treatment duration of 14 days, cardiac function and myocardial inflammation improved significantly compared to group D. Expression of CTLA-4, ICOS and ICOSL, B7-1 was statistically decreased in group A, B and C compared with group D. T-cell proliferation was inhibited by costimulatory blockade in a dose-dependent style. ICOSIg blockade significantly augments IL-4 and IL-10 production while diminished IFN-γ production.Conclusions: Blockade of costimulatory pathway with alliance therapy of CTLA4Ig and ICOSIg alleviated autoimmune damage in EAM and improved cardiac function. The mechanisms may be downregulation of costimulatory molecules and anti-inflammation.

Quantitative Assessment of Human β-Globin Gene Expression In Vitro by TaqMan Real-Time Reverse Transcription-PCR: Comparison with Competitive Reverse Transcription-PCR and Application to Mutations or Deletions in Noncoding Regions

β-Thalassemia is a genetic hemolytic disorder characterized by diminished production of β-globin chains, attributable to alterations within coding and noncoding sequences of the β-globin gene (1). Whether noncoding mutations are involved in the decrease of β-globin chain production or are simple polymorphisms remains a difficult issue (2). Although stable transfection with constructs bearing key regulatory β-locus control region elements upstream of the promoter are commonly used to assess the transcriptional effect of noncoding mutations in β-globin (3)(4)(5)(6)(7), discrepant results are still reported, as evidenced recently by the research team that used Northern blotting or ribonuclease protection assay to assess the functional impact of the +10 (-T) deletion (3)(5)(8). Although highly accurate, the competitive reverse transcription-PCR (RT-PCR) technique that we recently developed to quantify the transcriptional effect of genetic alterations in any part of the human β-globin gene is tedious and time-consuming (6). There is a need, therefore, for an expression method that is accurate, rapid, easier to perform, and well controlled quantitatively. In our current study, we developed a TaqMan® real-time quantitative RT-PCR assay (QRT-PCR), using the same in vitro expression model and the following human β-globin variants: −223T→C, −101C→T, −30T→A, +20C→T, IVS-I-108T→C, +10 (-T), and +15653→1577 (6). An additional …