Relative Real-time PCR Research Articles

Cryopreservation and long-term maintenance of bovine embryo-derived cell lines

The aim of this study was to develop methods for cryopreservation and long-term maintenance of putative bovine embryonic stem cells (ESCs). Putative bovine ESC (bESC) lines (n=3) isolated in conventional medium were used to compare slow-freezing and vitrification. After warming, vitrified cells (96.9%) demonstrated significantly (P<0.05) better survival than frozen-thawed cells (81.5%) and formed significantly more colonies with good morphology (vitrification: 93/93, 100.0%; slow-freezing: 74/106, 69.81%; P<0.05). The effect of inhibitors of differentiation (PD184352, SU5402, CHIR99021) on ESC maintenance was assessed on putative bESC lines established in N2B27-3i medium (n=8) or conventional medium (n=1) after culture over 30 passages (>240 days). All cell lines expressed ALP, SSEA1, SSEA4, OCT4, REX1 and SSEA1. OCT4 expression was confirmed by relative real-time PCR and was upregulated in early passages of putative bESCs cultured in N2B27-3i (2.9±0.89-fold higher at Passage (P) 2-4), whereas the converse was observed later (P22-26; 2.2±0.1-fold increase in conventional medium). Putative bESC lines isolated in N2B27-3i medium (n=3) or conventional medium (n=1) were vitrified at P18 and, after warming, were cultured for a further 12 passages. These cells survived vitrification and expressed OCT4, REX1, SSEA1, ALP, SSEA1 and SSEA4. These results demonstrate that putative bESC lines that express pluripotent markers can be cultured long term and retain expression of pluripotent markers after vitrification.

Cloning and Characterization of Bombyx mori Cyclophilin A

Cyclophilins are originally identified as cytosolic binding protein of the immunosuppressive drug cyclosporine A. They have an activity of peptidyl prolyl cis/trans-isomerases (PPIase), which may play important roles in protein folding, trafficking, assembly and cell signaling. In this study, we report the cloning and characterization of a Bombyx mori cyclophilin A (bCypA) cDNA. The full-length cDNA of bCypA consist of 947 nucleotides with a polyadenylation signal sequence AATAAA and contain an open reading frame of 498 nucleotides encoding a polypeptide of 166 amino acids. The deduced amino acid sequence of bCypA shares a central peptidyl prolyl cis/trans-isomerase and a cyclosporin-A-binding domain with other cyclophilin sequences. Relative quantification real-time (RT) PCR analysis shows that mRNA transcripts of bCypA are detected in all the investigated tissues and highest expression level in the skin of 3-day-old 5 instar larva. Also, bCypA had PPIase activity on the proline-containing peptides. Accordingly, we suggest that bCypA is a new member of the cyclophilin A (CyPA) family and will be useful for quality control of bioactivity recombinant proteins with proline-containing peptides.

Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs

BackgroundClassical swine fever (CSF), caused by the Classical swine fever virus (CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand the distribution, tissue tropism, pathogenesis, replication and excretion of CSFV in pigs. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of the experimentally infected pigs using real-time RT-PCR and immunohistochemistry (IHC).ResultsA relative quantification real-time PCR was established and used to detect the virus load in internal organs of the experimentally infected pigs. The study revealed that the virus was detected in all 21 of the internal organs and blood collected from pigs at day 1 to day 8 post infections, and had an increasing virus load from day 1 to day 8 post infections. However, there was irregular distribution virus load in most internal organs over the first 2 days post infection. Blood, lymphoid tissue, pancreas and ileum usually contain the highest viral loads, while heart, duodenum and brain show relatively low viral loads.ConclusionsAll the data suggest that CSFV had an increasing virus load from day 1 to day 8 post infections in experimentally infected pigs detected by real-time RT-PCR, which was in consistent with the result of the IHC staining. The data also show that CSFV was likely to reproduce in blood, lymphoid tissue, pancreas and the ileum, while unlikely to replicate in the heart, duodenum and brain. The results provide a foundation for further clarification of the pathogenic mechanism of CSFV in internal organs, and indicate that blood, lymphoid tissue, pancreas and ileum may be preferred sites of acute infection.

Construction and characterization of a cDNA library from shark regenerated hepatic tissue

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10 −5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10 −5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10 −5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.

Identification and Characterization of L1-Mediated Large Tandem Duplication, Spanning Exon 4 to 10 of the F13A1, In a Patient with Congenital Factor XIII Deficiency.

AbstractAbstract 3659Blood coagulation factor XIII (FXIII) is a plasma glycoprotein that plays an important role in the stabilization of fibrin clot in the final stage of blood coagulation. The FXIII circulates as a heterotetramer composed of two A and two B subunits in plasma. The A subunit (FXIIIA) possesses catalytic activity and this catalytic subunit is carried and protected by the B subunit (FXIIIB). Inherited deficiency of FXIII is a rare autosomal recessive bleeding disorder. Based on the genotype, it is classified into two types: FXIIIA deficiency (>95% of all cases), characterized by mutations in the F13A1 gene, and FXIIIB deficiency, characterized by mutations in the F13B gene. At the time of writing, at least 86 different mutations, most of which are point mutations, have been identified and registered in the F13-database. Here we show a novel, large tandem duplication in the F13A1 gene of a patient with congenital factor XIII deficiency. A female patient, born to consanguineous parents, suffered from severe bleeding diathesis, including menorrhagia, intracranial hemorrhage and ovarian hemorrhage, from childhood. Bleeding manifestations had been successfully controlled by monthly prophylactic replacement therapy using factor XIII concentrate (Fibrogammin). Trough levels of both factor XIII activity and antigen were 19% (Berichrom FXIII) and <10% (ELISA method), respectively. No pathogenic mutations associated with FXIII deficiency were detected from nucleotide sequencing of the coding region, 5′-UTR and 3′-UTR of both F13A1 and F13B. However, from an observation of the RT-PCR amplification state, the F13A1 mRNA level of the patient was apparently lower than that of healthy individuals. This result suggested the existence of abnormalities in the patient's F13A1. Relative exon copy number analysis using real-time PCR revealed two times as many of the continuing 7 exons (exon 4–10) as in the remaining region of the F13A1. This is likely due to some genomic rearrangement, probably homologous recombination in the F13A1 gene. IVS-3 and IVS-10 of F13A1 were very large and contained many repetitive sequences. The existence of an almost full-length (≂f6kb) L1 element, a well known long interspersed repetitive element (LINE) in humans, in both introns suggests that the recombination might arise from the L1 element. The provision of the PCR product (amplified by an IVS-10 specific forward primer and an IVS-3 specific reverse primer) confirmed that IVS-10 connected to IVS-3 with L1 as the boundary. Furthermore, sequencing this PCR product identified the 15bp sequence in the L1 element as an actual breakpoint. Taken together with the results of the analysis of the genomic DNA, this confirmed that the L1-mediated large (≂f109kb) tandem duplication was located in the patient's F13A1. In order to quantify the F13A1 mRNA, a relative real-time PCR quantification was performed using TaqMan Gene Expression Assays. Three different positions: one upstream of duplication (Exon2-3), one at duplication (Exon5-6) and one downstream of the duplication (Exon13-14) were used. The mRNA level of the patient was a markedly low compared to the normal control and the duplicated region (28% of normal) was clearly higher than both the upstream and downstream positions (9% of normal for each). This result reflected that the mRNA was probably maintaining the duplication. In order to analyze the mRNA splicing of the joint between the two duplicated regions, RT-PCR using a forward primer in exon 9 and a reverse primer in exon 4 of F13A1 was performed. Two major transcripts were amplified. The larger transcript was the product that keeps the genomic exon order and connects exon 10 to 4. However, the transcript is thought to lead to frameshift and to generate premature termination codon in exon 4. The other was the product that was skipping exon 10 and connected exon 9 to 4. This transcript is thought to escape frameshift and may translate to the unusual extra large FXIIIA. However, it is unlikely that the protein translated from the extra large mRNA circulates in blood. In conclusion, we identified an L1-mediated large tandem duplication, spanning exon 4 to 10 of the F13A1 gene, as an etiology of the congenital FXIII deficiency. Disclosures:No relevant conflicts of interest to declare.

P06 Hepatic lumican expression in paediatric non-alcoholic fatty liver disease

IntroductionLumican is a glycoprotein involved in collagen cross-linking and modulation of the innate immune system. Overexpression of lumican was recently described in a group of adult patients with histologically progressive...

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

Real-time reverse transcription–PCR expression profiling of porcine troponin I family in three different types of muscles during development

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.

Characterization of kinesin-like proteins in silkworm posterior silkgland cells

Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.

Cytochrome P450 aromatases: Impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus

Present study analyzed the importance of two forms of aromatases during ovarian development and recrudescence of north African/air-breathing catfish. We cloned both CYP19A1 (1941 bp; ovarian form) and CYP19A2 (1786 bp; brain form), which showed 47% homology between the two forms. Characterization of encoded proteins in non-steroidogenic COS-7 cells illustrated that both isoforms efficiently catalyzed the aromatization reaction by producing estradiol-17β (E 2) from testosterone. Tissue distribution pattern revealed preferential expression of CYP19A2 in brain while CYP19A1 predominated in ovary with trace amounts detected in other tissues including brain. Relative real-time PCR analysis revealed high transcript levels of both isoforms in the prespawning phase of ovarian cycle, which is in accordance with serum E 2 level. Aromatase activity in brain was comparatively lower than ovary, indicating the predominant requirement of aromatase in ovary. Ontogeny studies displayed sexual dimorphism, with early expression of CYP19A1 and CYP19A2 in ovary and brain, respectively. Phase-dependent rise of expression and enzyme activity of aromatase after hCG treatment revealed the stimulatory role of gonadotropin during preparatory and prespawning phases, preferentially to promote vitellogenesis. Lack of influence of hCG treatment during spawning phase endorses it further. A good correlation of expression, enzyme activity and serum E 2 levels suggests a crucial role of CYP19A1 during ovarian differentiation and ovarian cycle of catfish. Likewise, CYP19A2 might also be involved in these processes either indirectly or directly.

Expression of mast cell tryptase in scar

To investigate the expression and distribution of mast cell tryptase (MCT) in scar, and to discuss the different MCT gene expression in keloid, hypertrophic scar and normal skin. 20 samples of keloid, 20 samples of hypertrophic scar and 20 samples of normal skin were collected. The distribution of MCT was investigated by immunofluorescence histochemistry, and the MCT mRNA expression was detected by Relative Quantification real-time fluorescent PCR. MCT gene was mainly located in the collagen fiber bundles of the scar, especially in the superficial layer of scar. MCT mRNA expression was significantly higher in keloid than that in hypertrophic scar and normal skin (P < 0.01). Averagely, the MCT gene expression in keloid was 2.5 times and 5.4 times of that in hypertrophic scar and normal skin. MCT gene may play a role in the pathogenesis of scar.

54: Prevention of the alcohol-induced changes in brain-derived neurotrophic factor expression using neuroprotective peptides in a model of fetal alcohol syndrome

ObjectiveFetal alcohol syndrome (FAS) is the most common non genetic cause of mental retardation; prenatal alcohol exposure affects the development of the nervous system. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is essential in normal neuronal development and survival as well as synaptic plasticity, an important factor in learning and memory. Previously, we have shown that prenatal treatment with neuroprotective peptides, NAP+SAL, prevented alcohol induced fetal anomalies and learning deficits. Our objective was to evaluate if NAP+SAL′s neuroprotective effects are mediated by preventing alcohol-induced changes in BDNF expression during embryonic development.Study DesignA well-characterized FAS model was used (Webster, 1980). Timed, pregnant C57BL6/J mice were treated on gestational day 8 (E8) with alcohol (0.03 mL/g), placebo or alcohol+peptides (20g, 30 min before alcohol). Embryos were harvested at 6h(E8), 24h(E9) and day 10(E18). Each sample included at least 3 embryos, representing at least 3 litters per time point. Calibrator-normalized relative real-time PCR was performed to quantify BDNF with HPRT1 standardization. Statistics included ANOVA and Fisher PLSD.ResultsConclusionNAP+SAL prevented alcohol-induced changes in BDNF expression 24h and 10 days after alcohol exposure. This may explain, at least in part, the peptides prevention of neurodevelopmental anomalies and learning deficits in fetal alcohol syndrome. ObjectiveFetal alcohol syndrome (FAS) is the most common non genetic cause of mental retardation; prenatal alcohol exposure affects the development of the nervous system. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is essential in normal neuronal development and survival as well as synaptic plasticity, an important factor in learning and memory. Previously, we have shown that prenatal treatment with neuroprotective peptides, NAP+SAL, prevented alcohol induced fetal anomalies and learning deficits. Our objective was to evaluate if NAP+SAL′s neuroprotective effects are mediated by preventing alcohol-induced changes in BDNF expression during embryonic development. Fetal alcohol syndrome (FAS) is the most common non genetic cause of mental retardation; prenatal alcohol exposure affects the development of the nervous system. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is essential in normal neuronal development and survival as well as synaptic plasticity, an important factor in learning and memory. Previously, we have shown that prenatal treatment with neuroprotective peptides, NAP+SAL, prevented alcohol induced fetal anomalies and learning deficits. Our objective was to evaluate if NAP+SAL′s neuroprotective effects are mediated by preventing alcohol-induced changes in BDNF expression during embryonic development. Study DesignA well-characterized FAS model was used (Webster, 1980). Timed, pregnant C57BL6/J mice were treated on gestational day 8 (E8) with alcohol (0.03 mL/g), placebo or alcohol+peptides (20g, 30 min before alcohol). Embryos were harvested at 6h(E8), 24h(E9) and day 10(E18). Each sample included at least 3 embryos, representing at least 3 litters per time point. Calibrator-normalized relative real-time PCR was performed to quantify BDNF with HPRT1 standardization. Statistics included ANOVA and Fisher PLSD. A well-characterized FAS model was used (Webster, 1980). Timed, pregnant C57BL6/J mice were treated on gestational day 8 (E8) with alcohol (0.03 mL/g), placebo or alcohol+peptides (20g, 30 min before alcohol). Embryos were harvested at 6h(E8), 24h(E9) and day 10(E18). Each sample included at least 3 embryos, representing at least 3 litters per time point. Calibrator-normalized relative real-time PCR was performed to quantify BDNF with HPRT1 standardization. Statistics included ANOVA and Fisher PLSD. Results ConclusionNAP+SAL prevented alcohol-induced changes in BDNF expression 24h and 10 days after alcohol exposure. This may explain, at least in part, the peptides prevention of neurodevelopmental anomalies and learning deficits in fetal alcohol syndrome. NAP+SAL prevented alcohol-induced changes in BDNF expression 24h and 10 days after alcohol exposure. This may explain, at least in part, the peptides prevention of neurodevelopmental anomalies and learning deficits in fetal alcohol syndrome.

Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2

Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA − mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA − mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

Prostaglandin Synthesis Genes are Differentially Transcripted in Normal and Pyometra Endometria of Bitches

Pro-inflammatory stimuli, such as endotoxins released by Gram-negative bacteria, are potent stimulators of prostaglandin (PG) synthesis. The aim of this study was to evaluate the gene transcription pattern of PG synthesis enzymes in normal (anestrous, n = 6 and diestrous, n = 8) and pyometra (n = 7) endometria of bitches. Uteri were collected during routine ovariohysterectomy, processed for histopathological evaluation and uterine contents cultured. Gene transcription of COX-1, COX-2, mPGES-1 and PGF-synthase (PGFS) were evaluated by relative real-time PCR and normalized with the ribosomal protein L27 (RPL27) housekeeping gene. Normal uteri had no histological abnormalities and were negative for bacteriology. All pyometra uteri were hyperplasic and Escherichia coli was the only isolated bacterium. Except for COX-1, gene transcription was significantly higher in pyometra than in normal endometria. No significant differences in gene transcription were observed between normal diestrous and anestrous endometria. COX-2 gene transcription was 19 and 69 times higher in pyometra than in diestrous and anestrous endometria (p < 0.001), while PGFS gene transcription had a 3- and 600-fold increase in pyometra endometria compared to normal diestrous and anestrous endometria (p < 0.001). Gene transcription of mPGES-1 was 9 times higher in pyometra than in normal uteri (p < 0.01). Based on these results, we suggest that pyometra-associated E. coli endotoxin release stimulates the up-regulation of COX-2 PGFS and mPGES-1 gene transcription in the endometrium.

Erratum to: Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

This article unfortunately contained a mistake. The strain designations of two species, Prevotella brevis and Prevotella bryantii, were inadvertently reversed in Table 2. The names and sequences of the corresponding species-specific primers were not in error. The corrected table appears below.

Gene Transcription of TLR2, TLR4, and LPS Ligands in the Endometrium of the Bitch with Cystic Endometrial Hyperplasia-Pyometra Complex.

Cystic endometrial hyperplasia (CEH)-pyometra complex is the most frequent endometrial disorder in the bitch. Gram negative are the most frequent bacteria isolated in cases of pyometra and, their isolation are associated with more frequent and severe systemic signs. Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing the immune response to pathogens. TLR4 and TLR2 are recognized as receptors for components of Gram negative and Gram-positive bacteria (and lipoproteins of Gram-negative bacteria), respectively. Complex formation with bacterial co-receptor CD14 and lipoprotein binding protein (LBP), as well as MD-2 association, is necessary for maximal activation of TLR4 signaling. To the best of our knowledge, no data was reported on the expression of TLRs in the canine uterus. Therefore, the aim of this study was to evaluate the gene transcription of TLR2, TLR4 and LPS ligands in the endometrium of canine diestrous uteri without (n=7) or with pyometra (n=7). Uteri were collected during routine ovariohysterectomy (OVH) and uterine contents were cultured. Phase of the estrous cycle was determined by the observation of ovarian structures, recorded estrous date and measurement of plasma progesterone concentration by radioimmunoassay. Immediately after OVH, endometria were separated, rinsed with sterile RNAse free cold saline solution, immediately frozen in liquid nitrogen and then stored at −80°C until processed. For histological classification, according to De Bosscherre et al., (2001), tissue samples were fixed in 10% neutral phosphate buffered formalin. Gene transcription of TLR2, TLR4, CD14, MD2 and LBP were analyzed by relative real time PCR quantification and normalized with the housekeeping gene RPL27. All cases of pyometra were hyperplasic and E. coli was the only isolated bacteria. Diestrous normal uteri did not present signs of CEH and were negative for bacteriology. Gene transcription of TLR2 and TLR4 were respectively 8.3 and 2.4 times higher in pyometra cases compared to normal uteri (p< 0.001). Also, CD14, MD2 and LPB gene transcription showed a 7.4, 4.4 and 5.0 fold increase in pyometra cases compared to normal diestrous endometria, respectively (p< 0.001). This data shows that TLR2 and TLR4 are trancripted in the normal canine diestrous endometrium. It also shows that theses genes are up-regulated in E. coli pyometra cases. This up-regulation is probably ascribed to the stimulation by LPS and lipoprotein E. coli constituents. The stimulation by LPS is suggested by the observed up-regulation of CD14, LBP and MD2 transcription. (Supported by Grants POCTI/CVT/66587/2006 from Foundation for Science and Technology (FCT) and CIISA 74-Endometrial Hyperplasia. Maria Elisabete Silva was supported by a postdoctoral fellowship BPD/35031/2007 from FCT). *Mateus, L and Silva, E had equal contribution for this work. (poster)

Expression Levels of p53 Messenger RNA Detected by Real Time PCR in Tumor Tissue, Lymph Nodes and Peripheral Blood of Patients with Non-Small Cell Lung Cancer—New Perspectives for Clinicopathological Application

ABSTRACTThe importance of p53 mutations in the pathogenesis of lung cancer is well established, but it is still controversial whether the presence of p53 mutations or overexpression of P53 protein are of clinical significance. The aim of this study was to define the levels of expression of p53 mRNA in tumor, lymph nodes and peripheral blood of patients with non-small cell lung cancer, detected by relative quantification real-time PCR and to analyze the association with clinicopathological data. We applied real-time PCR to define the levels of expression of p53 mRNA in tumor, lymph nodes and blood of 25 patients with non-small cell lung cancer, which underwent an operation at the Department of Thoracic Surgery of the University Hospital for Pulmonary Diseases in Sofia. As a control group 25 healthy volunteers were used. The method of relative quantification was applied for the analysis. We detected a high expression of p53 mRNA in 90.9% of the tumors and in 73.7% of the lymph nodes. A trend for a correlation between the high levels of p53 expression in the tumor and lymph nodes was also established (p<0.19). We did not find any correlation between p53 expression in tumor tissue and peripheral blood.The expression analysis of p53 messenger RNA, detected by real-time PCR, can supplement the knowledge of p53 as a biomarker for lung cancer diagnosis and pathogenesis. The method is more sensitive than the currently used methods for p53 expression analysis and thus provides opportunities for a more accurate clinical application of molecular markers.

Another formula for calculating the gene change rate in real-time RT-PCR

Relative quantification real-time PCR has become a routine method in molecular biology research to study the small amount gene expression. There are a few mathematical models for relative quantification data analysis in real-time PCR, and the formula 2(-DDCT ) is one of the most frequently used method. In this paper, we are to present another equation which directly calculates the change rate (R (c)) of gene expression, and give a example to compare the application of this model with 2(-DDCT).

STAT3 Is Constitutively Phosphorylated on Serine 727 Residues, Activates Transcription, and Plays a Major Role in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in cellular physiology and organ development. Tyrosine phosphorylation of STAT3 provides cells with survival advantage and induces proliferation, whereas deletion of STAT3 results in embryonic lethality. As might be expected, STAT3 is commonly activated in neoplastic cells. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in a number of solid tumors and hematologic malignancies. A decade ago, Frank et al. (JCI 100:3149, 1997) reported that unlike other neoplasms in B-cell chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated (p) on serine 727 but not on tyrosine residues. This observation prompted us to investigate the role of STAT3 in CLL. We studied peripheral blood (PB) cells from ~100 patients with CLL using Western immunoblotting and found that in all patients, regardless of PB count, disease stage, or treatment status, STAT3 is constitutively phosphorylated on serine 727 but not on tyrosine residues. Serine pSTAT3 was detected in CD19+ but not in CD19− CLL PB-derived cells or in normal PB-derived B-lymphocytes isolated by immunomagnetic beads. Unlike constitutive serine pSTAT3, tyrosine pSTAT3 could be induced by incubating CLL cells with cytokines, such as interleukin (IL)-6, or anti-IgM antibodies. However, expression of cytokine-induced tyrosine pSTAT3 was transient and was no longer detected shortly (2 hours) after removal of the stimulating agent. In contrast, serine pSTAT3 levels remained stable in vitro for up to 72 hours, confirming that serine phosphorylation of STAT3 is constitutive in CLL cells. Because CLL is the only disease in which STAT3 is constitutively phosphorylated exclusively on serine residues and since tyrosine phosphorylation is thought to be required for STAT3 activation, we sought to determine whether serine pSTAST3 is biologically active. First, we isolated cytoplasmic and nuclear extracts and used Western immunoblotting to demonstrate that pSTAT3 is present both in the cytoplasm and the nucleus. Then, we confirmed these findings using confocal microscopy. After confirming that serine pSTAT3 translocates to the nucleus, we asked whether serine pSTAT3 binds to DNA. Using the electrophoretic mobility shift assay we demonstrated that nuclear STAT3 binds to a STAT3-specific DNA probe and that STAT3-DNA binding was eliminated by both anti-STAT3 and anti-serine pSTAT3 antibodies. Furthermore, although induction of tyrosine pSTAT3 by exogenous IL-6 slightly increased STAT3-DNA binding, complete dephosphorylation of tyrosine pSTAT3 by T-cell tyrosine phosphatase did not affect STAT3-DNA binding, suggesting that STAT3-DNA binding does not require tyrosine phosphorylation. Finally, pull-down of STAT3 with biotin-labeled DNA further confirmed that serine pSTAT3 binds to DNA in CLL cells. We then asked whether serine pSTAT3 initiates transcription. To answer this question we infected CLL cells with a lentiviral GFP-STAT3-shRNA and, as control, with GFP-empty vector and calculated changes in RNA levels by relative real-time PCR. In several CLL samples, infection with GFP-STAT3-shRNA induced a ~4-fold time-dependent reduction in the STAT3-regulated genes Bcl2, Pim1, Bcl-xL, Cyclin D1, p21 Waf1, and c-Myc, implying that serine pSTAT3 initiates transcription. Taken together, our data suggest that constitutive phosphorylation of STAT3 on serine 727 residues is a hallmark of CLL and induces activation of proliferation and survival genes. Therefore, STAT3 should be considered a therapeutic target in this disease.