Real-time RT-PCR Measurements Research Articles

Transcriptome – Scale characterization of salt responsive bean TCP transcription factors

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) proteins are important regulators of growth and developmental processes including branching, floral organ morphogenesis and leaf growth as well as stress response. This study identified 27 TCP genes of Phaseolus vulgaris (common bean), which were divided into three clusters based on phylogenetic relationship. In addition, this study showed that some of TCP genes such as Pvul-TCP-4 and Pvul-TCP-15 located on chromosomes 3 and 7, Pvul-TCP-7 and Pvul-TCP-20 located on chromosome 7 and 9, were segmentally duplicated. On the other hand, a total of 20 Pvul-TCP genes have predicted to be targeted by microRNAs (miRNA). Most of the miRNA-target genes were Pvul-TCP-1, -11, -13 and -27, which were targeted by 13, 17, 22 and 13 plant miRNAs, respectively. miR319 was one of the highly represented regulatory miRNAs to target TCP transcripts. Promoter region analysis of TCP genes resulted that the GT-1 motif, which was related to salt stress, was found in 14 different Pvul-TCP genes. Expression profiling of 10 Pvul-TCP genes based on RNA-sequencing data further confirmed with quantitative real-time RT-PCR measurements identified that Pvul-TCP genes under salt stress are expressed in a cultivar- and tissue-specific manner.

Pharmacological targeting of valosin containing protein (VCP) induces DNA damage and selectively kills canine lymphoma cells.

BackgroundValosin containing protein (VCP) is a critical mediator of protein homeostasis and may represent a valuable therapeutic target for several forms of cancer. Overexpression of VCP occurs in many cancers, and often in a manner correlating with malignancy and poor outcome. Here, we analyzed VCP expression in canine lymphoma and assessed its potential as a therapeutic target for this disease.MethodsVCP expression in canine lymphomas was evaluated by immunoblotting and immunohistochemistry. The canine lymphoma cell lines CLBL-1, 17–71 and CL-1 were treated with the VCP inhibitor Eeyarestatin 1 (EER-1) at varying concentrations and times and were assessed for viability by trypan blue exclusion, apoptosis by TUNEL and caspase activity assays, and proliferation by propidium iodide incorporation and FACS. The mechanism of EER-1 action was determined by immunoblotting and immunofluorescence analyses of Lys48 ubiquitin and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AFX). TRP53/ATM-dependent signaling pathway activity was assessed by immunoblotting for TRP53 and phospho-TRP53 and real-time RT-PCR measurement of Cdkn1a mRNA.ResultsVCP expression levels in canine B cell lymphomas were found to increase with grade. EER-1 treatment killed canine lymphoma cells preferentially over control peripheral blood mononuclear cells. EER-1 treatment of CLBL-1 cells was found to both induce apoptosis and cell cycle arrest in G1. Unexpectedly, EER-1 did not appear to act either by inducing ER stress or inhibiting the aggresome-autophagy pathway. Rather, a rapid and dramatic increase in γH2AFX expression was noted, indicating that EER-1 may act by promoting DNA damage accumulation. Increased TRP53 phosphorylation and Cdkn1a mRNA levels indicated an activation of the TRP53/ATM DNA damage response pathway in response to EER-1, likely contributing to the induction of apoptosis and cell cycle arrest.ConclusionsThese results correlate VCP expression with malignancy in canine B cell lymphoma. The selective activity of EER-1 against lymphoma cells suggests that VCP will represent a clinically useful therapeutic target for the treatment of lymphoma. We further suggest a mechanism of EER-1 action centered on the DNA repair response that may be of central importance for the design and characterization of VCP inhibitory compounds for therapeutic use.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1489-1) contains supplementary material, which is available to authorized users.

Expression of adenosine receptor subclasses in malignant and adjacent normal human prostate tissues.

Adenosine, a purine nucleoside plays important roles in the pathogenesis of cancer initiation and promotion via interaction with four adenosine receptors. In the present study we examined the differential expression pattern of adenosine receptors in the malignant and adjacent normal human prostate tissues. Prostate cancer tissue samples and adjacent normal tissues were obtained from 20 patients undergoing radical prostatectomy and histopathological diagnosis was confirmed for each sample. Total RNA was extracted and reverse transcribed into cDNA and the mRNA expression levels of adenosine receptors were investigated by Taq-man real-time RT-PCR experiment. Quantitative protein analysis was done by Western blotting experiment. Moreover, the mRNA and protein expression levels of adenosine receptors were measured after androgen treatment. Taq-man real-time RT-PCR measurements show different expression levels of adenosine receptor transcripts. A2B adenosine receptor was predominantly expressed in tumor tissues (2.4-fold) followed by significantly expression of A3 (1.6-fold) and A2A adenosine receptors (1.5-fold) compared to adjacent normal tissues. The presence of adenosine receptors at protein levels in prostate cancer tissues compared with normal tissues was shown the following rank order: A2B > A3 > A2A > A1 . Androgen receptor regulates adenosine receptors mRNA and protein expression in AR-positive LNCaP cells, which was not seen in AR-negative PC-3 cells. These results indicated for the first time, the differential mRNA expression profile and protein levels of adenosine receptors in the human prostate cancer. Interestingly, the A2B adenosine receptor followed by A3 is highly expressed in prostate tumor samples in comparison with the adjacent normal tissues. The findings support the possible key role of A2B adenosine receptor in promoting cancer cell growth and suggest that A2B may be a novel target for prostate cancer treatment.

Nrf2 deficiency impairs fracture healing in mice.

Oxidative stress plays an important role in wound healing but data relating oxidative stress to fracture healing are scarce. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that controls the cellular defence essential to combat oxidative stress by regulating the expression of antioxidative enzymes. This study examined the impact of Nrf2 on fracture healing using a standard closed femoral shaft fracture model in wild-type (WT) and Nrf2-knockout (Nrf2-KO)-mice. Healing was evaluated by histology, real-time RT-PCR, µCT and biomechanical measurements. We showed that Nrf2 expression is activated during fracture healing. Bone healing and remodelling were retarded in the Nrf2-KO compared to the WT-mice. Nrf2-KO-mice developed significantly less callus tissue compared to WT-mice. In addition, biomechanical testing demonstrated lower strength against shear stress in the Nrf2-KO-group compared to WT. The expression of vascular endothelial growth factor (VEGF) and osteocalcin is reduced during fracture healing in Nrf2-KO-mice. Taken together, our results demonstrate that Nrf2 deficiency in mice results in impaired fracture healing suggesting that Nrf2 plays an essential role in bone regeneration. Pharmacological activation of Nrf2 may have therapeutic potential for the enhancement of fracture healing.

Prognostic significance of PRAME expression based on immunohistochemistry for diffuse large B-cell lymphoma patients treated with R-CHOP therapy.

The preferentially expressed antigen of melanoma (PRAME), a tumor-associated antigen, is considered a prognostic marker for various human malignancies. The prognostic significance of PRAME expression for diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab-containing chemotherapy has not been evaluated to date, and the ability of immunohistochemistry (IHC) to detect PRAME expression in these patients has not yet been studied, although IHC is simple to perform in clinical practice. We evaluated the prognostic significance of PRAME expression based on IHC analysis in 160 DLBCL patients treated with R-CHOP therapy. There was a significant association between higher PRAME expression and shorter progression-free survival (PFS), and a trend toward shorter overall survival (OS) in patients with higher PRAME expression than that in patients with lower PRAME expression (5-year PFS, 48.1 vs. 61.1 %; 5-year OS, 65.6 vs. 79.1 %). Patients with high PRAME expression tended to have lower chemotherapeutic responses. Thus, IHC is useful for detecting and assessing PRAME expression in DLBCL. Further, we found a positive correlation between IHC and quantitative real-time RT-PCR measurements of PRAME expression. Our findings indicate that IHC results of PRAME expression can be a novel prognostic maker in DLBCL patients treated with R-CHOP therapy.

Effects of Glyceryl Glucoside on AQP3 Expression, Barrier Function and Hydration of Human Skin

Background/Aim: Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. Methods: In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. Results: AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. Conclusion: GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin.

Effect of electroacupuncture preconditioning at “Nèiguān” ( PC 6) on gene expression of myocardial opioid receptors in rats with ischemia-reperfusion

ObjectiveTo explore protective effects of electroacupuncture at “Nèiguān” (▪ PC 6) for preconditioning on myocardial ischemia-reperfusion injury (MIRI) and the mechanisms involved. MethodsForty-eight male Wistar rats were randomly divided into a sham operation group (Group N), a MIRI group (Group M) and an electroacupuncture (EA) group (Group E). The MIRI model was established by ligating the left anterior descending artery (LAD) for 30 min followed by reperfusion for 120 min. Partition sutures were passed under LAD without ligation for rats in Group N. Rats in Group E were pretreated with electroacupuncture (EA) applied at bilateral “Nèiguān” (▪ PC 6) for 20 min once a day for 3 consecutive days before ischemia. The infarct size plus the area at risk was evaluated by 2,3,5-triphenyltetrazolium chloride staining, and serum isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) levels were measured by biochemical methods. Myocardium morphological changes were observed under light microscopy. The mRNA expressions of myocardial δ and κ opioid receptors (DOR and KOR) were tested by real-time RT-PCR measurements. ResultsThe myocardial infarct size in Group E was more significantly decreased than that in Group M (P<0.05). The levels of CK-MB [(980 ± 92) U/L] and LDH [(2743 ± 124) U/L] in Group M were significantly higher than those in Group N [(312±41) U/L] and [(530 ± 56) U/L], respectively (both P<0.01). The levels of CK-MB [(572±70) U/L] and LDH [(1819±97) U/L] in Group E were significantly lower than those in Group M (both P<0.01). There were no significant differences in mRNA expressions of DOR and KOR between Group M and Group N (both P>0.05), but DOR expression in Group E was significantly higher than that either in Group M or in Group N (both P<0.01). No significant differences were found in KOR expression among the three groups (all P>0.05). ConclusionUp-regulation of expression of δ opioid receptors may be involved in protective effects of EA at Nèiguān (▪ PC 6) for preconditioning on MIRI.

Perioperative infliximab application has marginal effects on ischemia–reperfusion injury in experimental small bowel transplantation in rats

Ischemia-reperfusion injury leads to impaired smooth muscle function and inflammatory reactions after intestinal transplantation. In previous studies, infliximab has been shown to effectively protect allogenic intestinal grafts in the early phase after transplantation with resulting improved contractility. This study was designed to reveal protective effects of infliximab on ischemia-reperfusion injury in isogenic transplantation. Isogenic, orthotopic small bowel transplantation was performed in Lewis rats (3h cold ischemia). Five groups were defined: non-transplanted animals with no treatment (group 1), isogenic transplanted animals with vehicle treatment (groups 2/3) or with infliximab treatment (5mg/kg body weight intravenously, directly after reperfusion; groups 4/5). The treated animals were sacrificed after 3 (group 2/4) or 24h (group 3/5). Histological and immunohistochemical analysis, TUNEL staining, real-time RT-PCR, and contractility measurements in a standard organ bath were used for determination of ischemia-reperfusion injury. All transplanted animals showed reduced smooth muscle function, while no significant advantage of infliximab treatment was observed. Reduced infiltration of neutrophils was noted in the early phase in animals treated with infliximab. The structural integrity of the bowel and infiltration of ED1-positive monocytes and macrophages did not improve with infliximab treatment. At 3h after reperfusion, mRNA expression of interleukin (IL)-6, TNF-α, IL-10, and iNOS and MCP-1 displayed increased activation in the infliximab group. The protective effects of infliximab in the early phase after experimental small bowel transplantation seem to be unrelated to ischemia-reperfusion injury. The promising effects in allogenic transplantation indicate the need for further experiments with infliximab as complementary treatment under standard immunosuppressive therapy. Further experiments should focus on additional infliximab treatment in the setting of acute rejection.

Genetic polymorphisms in the formaldehyde dehydrogenase gene and their biological significance

The GSH-dependent formaldehyde dehydrogenase (FDH) is the most important enzyme for the metabolic inactivation of formaldehyde. We studied three polymorphisms of this gene with the intention to elucidate their relevance for inter-individual differences in the protection against the (geno-)toxicity of FA. The first polymorphism (rs11568816) was investigated using real-time PCR and restriction fragment analysis in 150 subjects. However, we did not find the polymorphic sequence in any of the subjects. We studied a second polymorphism (rs17028487), representing a base exchange (c.*114A>G) in exon 9 of the FDH gene. We analyzed 70 subjects with the SNaPshot Primer Extension method and subsequent analysis in a ABI PRISM 3100, but no variant allele was identified. A third polymorphism, rs13832 in exon 9 (c.*493G>T), was studied in a group of 105 subjects by the SNaPshot Primer Extension method. 43 of the subjects were heterozygous for the polymorphism (G/T), 46 homozygous for the T allele, and 16 were homozygous for the G-allele. Real-time RT-PCR measurements of FDH mRNA did not indicate a significant difference in transcript levels between the heterozygous and the homozygous groups. The in vitro comet assay after FA exposure of blood samples obtained from 5 homozygous GG and 3 homozygous TT subjects did not lead to a significant difference between these two groups. Altogether, our study did not identify biologically relevant polymorphisms in transcribed regions of the FDH gene, which may lead to inter-individual differences in the metabolic inactivation of FA.

Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways

Baicalin is an important medicinal herb purified from the dry roots of Scutellaria baicalensis Georgi. The present study was undertaken to evaluate the neuroprotective effects of baicalin in gerbils subjected to transient global cerebral ischemic–reperfusion injury. Baicalin at doses of 50, 100 and 200mg/kg was intraperitoneally injected into the gerbils immediately after cerebral ischemia. Seven days after reperfusion, hematoxylin and eosin (HE) staining was performed to analyze hippocampal CA1 pyramidal damage histopathologically. In addition, in order to understand the potential protective mechanism of baicalin, we examined anti-oxidative enzymes, such superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), non-enzymatic scavenger glutathione (GSH) and measured the content of malondialdehyde (MDA) in hippocampus. The mRNA and protein expressions of BDNF were determined in ischemic hippocampus by real-time RT-PCR and Western blot, respectively. Evidence for neuronal apoptosis was detected by real-time RT-PCR, Western blot and caspase-3 activity measurement. Histopathological examination showed that the administration of baicalin by the dose of 100 and 200mg/kg significantly attenuated ischemia-induced neuronal cell damage. Reduced level of MDA, obviously elevated activities of SOD and GSH as well as GSH-PX were also found in baicalin-treated groups. Further investigation demonstrated that treatment with baicalin remarkably promoted the expression of BDNF and inhibited the expression of caspase-3 at mRNA and protein levels by real-time RT-PCR and Western blot, respectively. Besides, caspase-3 activity assay also elucidated that the administration of baicalin could significantly suppress caspase-3 in ischemic gerbils hippocampus. Theses findings suggest that baicalin's neuroprotection appears to be associated with its anti-oxidative and anti-apoptotic properties in global cerebral ischemia in the gerbils.

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

BackgroundRecent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.MethodsUsing a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined.ResultsWe show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.ConclusionsWe propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.

In VivoRole of Platelet-Derived Growth Factor–BB in Airway Smooth Muscle Proliferation in Mouse Lung

Airway smooth muscle (ASM) hyperplasia in asthma likely contributes considerably to functional changes. Investigating the mechanisms behind proliferation of these cells may lead to therapeutic benefit. Platelet-derived growth factor (PDGF)-BB is a well known ASM mitogen in vitro but has yet to be directly explored using in vivo mouse models in the context of ASM proliferation and airway responsiveness. To determine the in vivo influence of PDGF-BB on gene transcripts encoding contractile proteins, ASM proliferation, and airway physiology, we used an adenovirus overexpression system and a model of chronic allergen exposure. We used adenovirus technology to selectively overexpress PDGF-BB in the airway epithelium of mice. Outcome measurements, including airway physiology, real time RT-PCR measurements, proliferating cell nuclear antigen staining, and airway smooth muscle quantification, were performed 7 days after exposure. The same outcome measurements were performed 24 hours and 4 weeks after a chronic allergen exposure model. PDGF-BB overexpression resulted in airway hyperresponsiveness, decreased lung compliance, increased airway smooth muscle cell numbers, positive proliferating cell nuclear antigen-stained airway smooth muscle cells, and a reduction in genes encoding contractile proteins. Chronic allergen exposure resulted in elevations in lung lavage PDGF-BB, which were observed in conjunction with changes in gene transcript expression encoding contractile proteins and ASM proliferation. We demonstrate for the first time in vivo that PDGF-BB induces ASM hyperplasia and changes in lung mechanics in mice and that, during periods of allergen exposure changes in lung, PDGF-BB are associated with changes in airway structure and function.

Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors

Clostridium thermocellum produces a highly efficient cellulolytic extracellular complex, termed the cellulosome, for hydrolyzing plant cell wall biomass. The composition of the cellulosome is affected by the presence of extracellular polysaccharides; however, the regulatory mechanism is unknown. Recently, we have identified in C. thermocellum a set of putative σ and anti-σ factors that include extracellular polysaccharide-sensing components [Kahel-Raifer et al. (2010) FEMS Microbiol Lett 308:84-93]. These factor-encoding genes are homologous to the Bacillus subtilis bicistronic operon sigI-rsgI, which encodes for an alternative σ(I) factor and its cognate anti-σ(I) regulator RsgI that is functionally regulated by an extracytoplasmic signal. In this study, the binding of C. thermocellum putative anti-σ(I) factors to their corresponding σ factors was measured, demonstrating binding specificity and dissociation constants in the range of 0.02 to 1 μM. Quantitative real-time RT-PCR measurements revealed three- to 30-fold up-expression of the alternative σ factor genes in the presence of cellulose and xylan, thus connecting their expression to direct detection of their extracellular polysaccharide substrates. Cellulosomal genes that are putatively regulated by two of these σ factors, σ(I1) or σ(I6), were identified based on the sequence similarity of their promoters. The ability of σ(I1) to direct transcription from the sigI1 promoter and from the promoter of celS (encodes the family 48 cellulase) was demonstrated in vitro by runoff transcription assays. Taken together, the results reveal a regulatory mechanism in which alternative σ factors are involved in regulating the cellulosomal genes via an external carbohydrate-sensing mechanism.

The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

Amylin mRNA in the rat brain: induction of its expression in the preoptic area of rat dams

Event Abstract Back to Event Amylin mRNA in the rat brain: induction of its expression in the preoptic area of rat dams Arpad Dobolyi1* 1 Hungarian Academy of Sciences and Semmelweis University, Department of Anatomy, Hungary Postpartum behavioral and neuroendocrine changes in females are critically important parts of mammalian reproduction and are expected to be associated with gene expressional alterations. The preoptic area of the hypothalamus plays a central role in postpartum regulations as concluded from the absence of maternal behaviors following preoptic lesions in rats. In the present study, a microarray experiment was performed to investigate gene expressional regulations in rat dams. Preoptic mRNA levels of 4 lactating dams and 4 mothers whose pups were removed on the day of delivery were compared 9 days after parturition. We found 95 genes whose expression increased and 96 genes whose expression decreased significantly. The gene whose expression level demonstrated the largest change, a 25.7 times increase, was amylin, or islet amyloid polypeptide. Subsequently, quantitative real-time RT-PCR measurements were performed using mRNA from the preoptic area of 12 lactating dams and 12 mothers whose pups were removed. A 24.1 times, highly significant increase in the mRNA level of amylin validated the induction of its expression found by the microarray experiment. In situ hybridization histochemistry for amylin demonstrated its expression in the medial preoptic nucleus and parts of the lateral preoptic area but nowhere else in the rat brain. Amylin is a 37 aa peptide previously described only in the pancreas. In this study, we first revealed its expression in the central nervous system and also showed that the level of amylin expression is very low in control female rats and in mothers deprived of their pups but is dramatically induced in normal mothers. The results suggest a role for amylin in the control of physiological and behavioral alterations in mother rats. Support was provided by the Hungarian Science Foundation NKTH-OTKA K67646 grant. Arpád Dobolyi is grantee of the Bolyai János Scholarship. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Homeostatic regulatory mechanisms Citation: Dobolyi A (2009). Amylin mRNA in the rat brain: induction of its expression in the preoptic area of rat dams. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.008 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Feb 2009; Published Online: 25 Feb 2009. * Correspondence: Arpad Dobolyi, Hungarian Academy of Sciences and Semmelweis University, Department of Anatomy, Budapest, Hungary, dobolyi@ana.sote.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Arpad Dobolyi Google Arpad Dobolyi Google Scholar Arpad Dobolyi PubMed Arpad Dobolyi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The extremely halophilic archaeon Halobacterium salinarum R1 responds to potassium limitation by expression of the K+-transporting KdpFABC P-type ATPase and by a decrease in intracellular K+

Halobacterium species balance high external osmolality by the accumulation of almost equimolar amounts of KCl. Thus, steady K(+) supply is a vital prerequisite for life of these extreme halophiles. So far, K(+) is reported to enter the halobacterial cell only passively by use of potential-driven uniporters. However, the genome of both the extreme halophilic archaeon Halobacterium sp. NRC-1 and H. salinarum R1 comprises one single gene cluster containing the genes kdpFABC coding for homologs of the bacterial ATP-driven K(+) uptake system KdpFABC, together with an additional ORF so far annotated as cat3 in Halobacterium sp. NRC-1 and as UspA protein in H. salinarum R1 (the ORF is only referred to as cat3 in the following). Deletion of the kdpFABCcat3 genes led to a reduced ability to grow under limiting K(+) concentrations, whereas real-time RT-PCR measurements revealed cat3-dependent high expression rates of the Kdp system in case of external K(+) depletion. Synthesis of the KdpFABC complex enables H. salinarum R1 to grow under extreme potassium-limiting conditions of >20 microM K(+). These results provide the first experimental evidence of an ATP-driven K(+) uptake system in Halobacterium. Moreover, H. salinarum R1 was shown to further adapt to K(+) limitation by a significant decrease of the intracellular K(+) level, which suggests a rather complex mechanism of K(+) homeostasis, in which the adaptation of cellular K(+) concentrations and the concomitant transcriptional regulation of genes coding for a high-affinity ATP-driven K(+) uptake system ensure the essential potassium supply under limiting conditions.

Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes

Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.

Quantitative real-time RT-PCR measurement of cytokine mRNA expression in the skin of normal cats and cats with allergic skin disease

Feline allergic skin disease is thought to be associated with dermal infiltration of Th 2 lymphocytes and synthesis of associated cytokines. In this study, real-time RT-PCR assays were developed to measure feline interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the skin of healthy control cats, and in the lesional and non-lesional skin of cats with allergic skin disease. Total RNA was extracted from skin biopsies using the RNeasy Mini Kit with on-column and in-solution DNase digestion steps. cDNA was synthesised using Improm-II reverse transcriptase and random hexamers. Real-time PCR was carried out using an iCycler IQ system (Bio-Rad), and gene-specific primers were designed to span an exon/exon junction of each cytokine gene. Taq-man probes were used to add specificity to the system. Messenger RNA from the housekeeping gene GAPDH was used for normalisation of the cytokine threshold cycle. The eleven cytokine mRNA transcripts quantified were present at varying levels, but there was no apparent difference in expression between normal, non-lesional and lesional skin. TGF-β represented the most abundant transcript while IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 and TNF-α were present at levels approximately 1000-fold less. IL-2 and INF-γ represented the least abundant templates with no detectable copies in most RNA samples. This quantitative analysis of cytokine mRNA expression in feline skin biopsies has suggested that there is not a simple Th 2 bias in lesional skin of cats with allergic dermatopathies.

Activity‐dependent Gene Expression Correlates with Interictal Spiking in Human Neocortical Epilepsy

Interictal spikes are hallmarks of epileptic neocortex that are used commonly in both EEG and electrocorticography (ECoG) to localize epileptic brain regions. Despite their prevalence, the exact relationship between interictal spiking and the molecular pathways that drive the production and propagation of seizures is not known. We have recently identified a common group of genes induced in human epileptic foci, including EGR1, EGR2, c-fos, and MKP-3. We found that the expression levels of these genes correlate precisely with the frequency of interictal activity and can thus serve as markers of epileptic activity. Here, we explore this further by comparing the expression of these genes within human epileptic neocortex to both ictal and specific electrical parameters of interictal spiking from subdural recordings prior to surgical resection in order to determine the electrical properties of the human neocortex that correlate best to the expression of these genes. Seizure frequency as well as quantitative electrophysiological parameters of interictal spikes including frequency, amplitude, duration, and area were calculated at each electrode channel and compared to quantitative real-time RT-PCR measurements of four activity-dependent genes (c-fos, EGR1, EGR2, and MKP-3) in the underlying neocortical tissue. Local neocortical regions of seizure onset had consistently higher spike firing frequencies and higher spike amplitudes compared to nearby "control" cortex. In contrast, spike duration was not significantly different between these two areas. There was no relationship observed between seizure frequency and the expression levels of activity-dependent genes for the patients examined in this study. However, within each patient, there were highly significant correlations between the expression of three of these genes (c-fos, EGR1, EGR2) and the frequency, amplitude, and total area of the interictal spikes at individual electrodes. We conclude that interictal spiking is closely associated with the expression of a group of activity-dependent transcription factors in neocortical human epilepsy. Since there was little correlation between gene expression and seizure frequency, our results suggest that interictal spiking is a stronger driving force behind these activity-dependent gene changes and may thus participate in the development and maintenance of the abnormal neuronal hyperactivity seen in human epileptic neocortex.

Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR

Viral hemorrhagic septicemia virus (VHSV) is one of the most ubiquitous viruses in salmonid aquaculture in Europe. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Thus, accurate methods to test new antiviral compounds need to be developed. Our goal was to establish a model system for testing novel antivirals with potential applications to aquaculture. In a previous study, a TaqMan ® real-time RT-PCR assay was designed to detect and quantitate VHSV in rainbow trout tissues [Chico, V., Gomez, N., Estepa, A., Perez, L., 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132, 154–159]. In this report, we applied the real-time RT-PCR assay to the evaluation of the inhibitory effect of ribavirin, a well-known broad spectrum antiviral drug, in a cell culture system. When added from the beginning of the infection, ribavirin caused a dose-dependent reduction of VHSV RNA accumulation. Real-time RT-PCR measurements showed 99.8% inhibition at 25 μg/ml ribavirin, with an IC50 of 0.43 μg/ml. Ribavirin maintained its inhibitory activity against VHSV when added at 6 h post-infection. Quantitation of N protein messenger RNA and plus-stranded RNA showed a substantial decrease of viral transcription in ribavirin-treated cells. Partial reversion of the effect of ribavirin by addition of GTP was observed, confirming that ribavirin targets the synthesis of guanidine nucleotides in the cells. This is the first report of a real-time PCR-based assay for addressing the efficacy and mechanism of action of an antiviral agent for rainbow trout.