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Quantitative Real-time Polymerase Chain Reaction (quantitative + real-time_polymerase_chain_reaction)
Selected AbstractsIdentification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cellsDEVELOPMENTAL DYNAMICS, Issue 2 2008Latasha C. Redmond Abstract Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin - 1, and muscleblind - like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis. Developmental Dynamics 237:436,446, 2008. © 2008 Wiley-Liss, Inc. [source] Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2009Sebastian Kreuz Abstract Background Acetyl-CoA carboxylases (ACC) 1 and 2 are central enzymes in lipid metabolism. To further investigate their relevance for the development of obesity and type 2 diabetes, expression of both ACC isoforms was analyzed in obese fa/fa Zucker fatty and Zucker diabetic fatty rats at different ages in comparison to Zucker lean controls. Methods ACC1 and ACC2 transcript levels were measured by quantitative real-time polymerase chain reaction in metabolically relevant tissues of Zucker fatty, Zucker diabetic fatty and Zucker lean control animals. Quantitative real-time polymerase chain reaction was also applied to measure ACC tissue distribution in human tissues. For confirmation on a protein level, quantitative mass spectrometry was used. Results Disease-related transcriptional changes of both ACC isoforms were observed in various tissues of Zucker fatty and Zucker diabetic fatty rats including liver, pancreas and muscle. Changes were most prominent in oxidative tissues of diabetic rats, where ACC2 was significantly increased and ACC1 was reduced compared with Zucker lean control animals. A comparison of the overall tissue distribution of both ACC isoforms in humans and rats surprisingly revealed strong differences. While in rats ACC1 was mainly expressed in lipogenic and ACC2 in oxidative tissues, ACC2 was predominant in oxidative and lipogenic tissues in humans. Conclusion Our data support a potential role for both ACC isoforms in the development of obesity and diabetes in rats. However, the finding of fundamental species differences in ACC1 and ACC2 tissue expression might be indicative for different functions of both isoforms in humans and rats and raises the question to which degree these models are predictive for the physiology and pathophysiology of lipid metabolism in humans. Copyright © 2009 John Wiley & Sons, Ltd. [source] Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zincENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Véronique Marie Abstract Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 ,M), Zn (15.3 ,M), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (106 times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level. [source] Molecular characterization of two novel esterase genes from carmine spider mite, Tetranychus cinnabarinus (Acarina: Tetranychidae)INSECT SCIENCE, Issue 2 2010Wei Sun Abstract, Two novel esterase complementary DNAs were identified and cloned from the insecticide-susceptible strain of Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae), which were designated as TCE1 and TCE2, respectively. The cDNA of TCE1 gene contained an open reading frame (ORF) of 1701 bp encoding 567 amino acids, and a predicted molecular weight of 62.75 kDa, the cDNA of TCE2 contained an ORF of 1680 bp encoding 560 amino acids, and a predicted molecular weight of 63.14 kDa. TCE1 and TCE2 were submitted to GenBank, accession number EU130461 and EU130462. The well-conserved sequence motif, GXSXG, used as a signature pattern in the esterase family are present in both TCE1 and TCE2 (GQSAG in TCE1, whereas GESAG in TCE2), indicating that these two genes are predicted to be esterases. Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from Boophilus microplus showed that TCE1 shares 33.98% identity and TCE2 shares 33.46% identity. TCE1 and TCE2 share 46.4% identity. Quantitative real-time polymerase chain reaction revealed that expression level of the TCE2 gene was relatively higher than that of the TCE1 in all instars examined except the protonymph, and the expression level of these two esterase genes in adults of T. cinnabarinus was significantly higher than that in any other instars, respectively. T. cinnabarinus is an important agricultural mite pest and esterases are important in the metabolisms of insects and mites; the genomic information obtained in this study will contribute to esterase molecular biological study on mite pest species. [source] Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2003Chisa Hidaka Background: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. Methods: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. Results: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. Conclusions: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. Clinical relevance: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell developmentNEW PHYTOLOGIST, Issue 3 2007Hong-Bin Li Summary ,,Reactive oxygen species (ROS) play important roles in multiple physiological processes such as cellular signalling and stress responses, whereas, the hydrogen peroxide (H2O2) scavenging enzyme ascorbate peroxidase (APX) participates in the regulation of intracellular ROS levels. ,,Here, a cotton (Gossypium hirsutum) cytosolic APX1 (GhAPX1) was identified to be highly accumulated during cotton fibre elongation by proteomic analysis. GhAPX1 cDNA contained an open reading frame of 753-bp encoding a protein of 250 amino acid residues. When GhAPX1 was expressed in Escherichia coli, the purified GhAPX1 was a dimer consisting of two identical subunits with a molecular mass of 28 kDa. GhAPX1 showed the highest substrate specificity for ascorbate. ,,Quantitative real-time polymerase chain reaction (PCR) analyses showed that GhAPX1 was highly expressed in wild-type 5-d postanthesis fibres with much lower transcript levels in the fuzzless-lintless mutant ovules. Treating in vitro cultured wild-type cotton ovules with exogenous H2O2 or ethylene induced the expression of GhAPX1 and hence increased total APX activity proportionally, followed by extended fibre cell elongation. ,,These data suggest that GhAPX1 expression is upregulated in response to an increase in cellular H2O2 and ethylene. GhAPX1 encodes a functional enzyme that is involved in hydrogen peroxide homeostasis during cotton fibre development. [source] Gene expression profiles of TNF-,, TACE, furin, IL-1, and matrilysin in UVA- and UVB-irradiated HaCat cellsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 4 2005Beata Skiba Background/Purpose: It is known that solar ultraviolet (UV) irradiation exerts multiple effects on mammalian skin tissues, one of which is the induction of local and systemic immunosuppression as well as inflammation. Tumor necrosis factor-, (TNF-,) and other cytokines are suggested to play a role in these responses. Quantitative real-time polymerase chain reaction (TaqMan RTPCR) was used to elucidate the effect of UVA and UVB irradiation on the expression of genes coding for TNF-,, IL-1,, IL-10, FasL, matrilysin, TACE and furin in HaCaT cells over a 48 h period (IL-1,, interleukin-1,; FasL, Fas ligand). Methods: Cultured HaCaT cells were either sham irradiated (control) or exposed to UVA (2000 and 8000 J/m2) or UVB (200 and 2000 J/m2) radiation. RNA was extracted from cells at 0, 4, 8, 12, 16, 24, 48 h post-irradiation and reverse transcribed to generate cDNA for subsequent real-time PCR amplification. Results: Significant increases in the mRNA levels for all genes tested were detected in both UVA- and UVB-irradiated HaCaT cells compared with control (sham-irradiated) cells. TNF-, mRNA levels were immediately up-regulated (0 h) after irradiation, with maximal induction at 8 h post 2000 J/m2 UVA and 200 J/m2 UVB irradiation, at 4 h post 8000 J UVA irradiation and at 48 h post 2000 J/m2 UVB irradiation. No correlation was observed between TNF-,, TACE and furin mRNA induction in the different irradiated cohorts. Conclusion: Results suggest that time-distinct gene induction of TNF-,, furin, IL-1, and matrilysin may be involved in UV-induced cellular responses, but not for TACE. In general, mRNA induction was dose dependent at some time points post-irradiation, but not throughout the whole time course tested. Our results show that quantitative real-time PCR is a useful tool in the analysis of quantitative changes of mRNA levels in cultured HaCaT cells after UV exposure. [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] The Role of Opiorphins (Endogenous Neutral Endopeptidase Inhibitors) in Urogenital Smooth Muscle BiologyTHE JOURNAL OF SEXUAL MEDICINE, Issue S3 2009Kelvin Paul Davies BSc ABSTRACT Introduction., The opiorphins are a newly characterized class of peptides that act as potent endogenous neutral endopeptidase (NEP) inhibitors. Recent reports have suggested that they play an important role in erectile physiology. Aim., This article reviews recent developments that increase our understanding of the role of the opiorphin family of peptides in erectile physiology. Methods., During a microarray screen of gene changes that occur in a rat diabetic model of erectile dysfunction (ED), Vcsa1 was one of the most down-regulated genes in the rat corpora. Quantitative real-time polymerase chain reaction demonstrated that in at least three models of diseases that result in ED (diabetes, aging, and cavernous nerve [CN] transection), Vcsa1 was down-regulated in the rat corpora. The human opiorphin family of genes (hSMR3A/B and ProL1) also acts as markers of erectile function in patients with ED. Main Outcome Measures., The reader will be informed of the most current research regarding the role of opiorphins in urogenital smooth muscle biology. Results., These observations led to the suggestion that genes encoding opiorphins (and potentially their peptide products) can act as markers of ED. Gene transfer of plasmids overexpressing Vcsa1 in aging rats, as well as intracorporal injection of sialorphin, led to an improvement in erectile function. In organ bath studies, we demonstrated that sialorphin can cause increased rates of relaxation of corporal smooth muscle (CSM). We have also demonstrated that in vitro, Vcsa1 causes changes in the expression of G-protein-coupled receptors (GPCRs). This has led us to suggest that the action of Vcsa1 on erectile physiology may act through relaxation of CSM by its ability to act as an inhibitor of NEP, therefore prolonging the action of peptide agonists at their GPCRs. Conclusions., Overall, there is a growing body of evidence that the opiorphins play a role in regulating CSM tone and thereby erectile function. Davies KP. The role of opiorphins (endogenous neutral endopeptidase inhibitors) in urogenital smooth muscle biology. J Sex Med 2009;6(suppl 3):286,291. [source] Vestibular Schwannoma Quantitative Polymerase Chain Reaction Expression of Estrogen and Progesterone Receptors,THE LARYNGOSCOPE, Issue 8 2008Andrew K. Patel MD Abstract Objectives/Hypothesis: Determine the role of estrogen receptor (ER) and progesterone receptor (PR) expression in sporadic and neurofibromatosis 2 (NF2)-related vestibular schwannomas (VS). Growth and proliferation signaling in human VS tumorigenesis may play a key role in molecular therapeutic targeting. VS carry mutations of the NF2 gene encoding the tumor suppressor, merlin, which interacts with ErbB2 in Schwann cells, implicating ErbB receptors in VS tumorigenesis. ErbB receptor family members are overexpressed or constitutively activated in many human tumors, and are effective therapeutic targets in some human cancers. VS occur more frequently in women and are larger, more vascular, and demonstrate increased growth rates during pregnancy. ER and PR may play a role in ErbB pathway activation and VS progression. Study Design: Quantitative real-time polymerase chain reaction (qRT-PCR) for ER and PR messenger RNA was performed using greater auricular and vestibular nerve controls (n = 8), sporadic VS (n = 23), and NF2-related VS (n = 16) tissues. Methods: The qRT-PCR data were normalized with standardization to a single constitutively expressed control gene, human cyclophylin. Results: Reverse transcription of messenger RNA from control and tumor specimens followed by RT Q-PCR demonstrated differences in ER and PR gene expression between sporadic and NF2-related VS. Conclusions: ER and PR expression in VS might have implications for development of a VS-specific drug delivery system using antihormone and ErbB pathway small molecule inhibitors, due to crosstalk between these receptors. These signals may be critical for re-establishing ErbB-mediated cell density dependent growth inhibition. [source] Impaired lymphangiogenesis due to excess vascular endothelial growth factor-D/Flt-4 signalling in the skin of patients with systemic sclerosisBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2010N. Honda Summary Background, Vascular abnormalities are one of the primary pathological components of systemic sclerosis (SSc). However, it has not been determined if there are also abnormalities in the formation of lymphatic vessels in SSc. Objective, To evaluate lymphangiogenic activity in SSc skin. Methods, The numbers of D2-40-positive lymphatic vessels in skin specimens from healthy control subjects and patients with SSc were counted and compared. Quantitative real-time polymerase chain reaction (PCR) was performed to determine mRNA levels of vascular endothelial growth factor (VEGF)-D and Flt-4 (fms-related tyrosine kinase 4, VEGFR-3, one of the receptors for VEGF-D) in the skin. Serum VEGF-D levels were measured with specific enzyme-linked immunosorbent assays. Results, The number of lymphatic vessels in patients with SSc was significantly decreased compared with healthy control subjects. Mean relative transcript levels of FIGF (VEGF-D) and FLT4 (Flt-4) in skin tissue from patients with SSc were significantly increased compared with healthy control subjects. By the analysis of the association between serum VEGF-D levels and the clinical or laboratory features, we found that patients with SSc with higher serum VEGF-D levels more frequently have skin ulcers than those with normal VEGF-D levels. Conclusions, A systemic increase of VEGF-D, as well as local overexpression of FIGF and FLT4, may be the cause of disturbed lymphangiogenesis in SSc skin and play a role in the pathogenesis of SSc. We showed the possibility that regulation of VEGF-D/Flt-4 signalling could lead to new treatment of skin ulcers in SSc by controlling the formation of lymphatic vessels. [source] Dominant expression of interleukin 10 but not interferon , in CD4,CD8,,,T cells of autoimmune lymphoproliferative syndromeBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2002Shouichi Ohga Summary. Cytokine expression in CD4,CD8, double-negative (DN) T cells of autoimmune lymphoproliferative syndrome (ALPS) was analysed. Two patients with DN ,,T-cell expansion showed higher serum interleukin 10 (IL-10) levels than one patient without it. Intracellular flow-cytometric analysis indicated the IL-10-expressing CD3+CD4,CD8, cells in patients with lymphoproliferation. Quantitative real-time polymerase chain reaction revealed ,100 times higher IL-10, but not interferon-, or transforming growth factor-, in DN than in single-positive T cells. IL-10 was exclusively expressed in DN ,, but not ,,, cells. Circulating DN ,,T cells may constitutively express IL-10 and contribute to the ALPS phenotype. [source] COPS3 amplification and clinical outcome in osteosarcomaCANCER, Issue 9 2007Taiqiang Yan MD Abstract BACKGROUND. Amplification of several genes that map to a region of chromosome 17p11.2, including COPS3, was observed in high-grade osteosarcoma. These genes were also shown to be overexpressed and may be involved in osteosarcoma tumorigenesis. COPS3 encodes a subunit of the COP9 signalosome implicated in the ubiquitination and ultimately degradation of the P53 tumor suppressor. To determine the relation between COPS3 amplification, P53 mutation, and patient outcome in osteosarcoma, tumors from a large cohort of patients with high-grade osteosarcoma and long-term clinical follow-up were examined. METHODS. Quantitative real-time polymerase chain reaction (PCR) was performed to detect copy number changes for COPS3, as well as additional genes (NCOR1, TOM1L2, and PMP22) from the 17p11.2 amplicon, in 155 osteosarcomas from a prospective collection of tumors with corresponding clinical data. Univariate and multivariate analyses were performed to assess differences in survival between groups. RESULTS. Amplification of COPS3, detected in 31% of the osteosarcomas, was strongly associated with large tumor size (P = .0009), but was not associated with age at diagnosis, site, sex, and tumor necrosis. COPS3 amplification was significantly correlated with a shorter time to metastasis with an estimated hazard ratio (HR) of 1.61 (95% confidence interval [CI], 1.02,2.55) in univariate analysis (log-rank test, P = .042). However, in an a priori multivariate Cox model including the other clinical parameters, the HR for COPS3 amplification decreased to 1.32 (95% CI, 0.82,2.13, P = .25), mainly due to the strong correlation with tumor size. COPS3 amplification and P53 mutation frequently occurred in the same tumors, suggesting that these are not mutually exclusive events in osteosarcoma. Although not statistically significant, patients whose tumors exhibited both molecular alterations tended to be more likely to develop metastasis compared with patients with either COPS3 amplification or P53 mutation alone. CONCLUSIONS. COPS3 is the likely target of the 17p11.2 amplicon. COPS3 may function as an oncogene in osteosarcoma, and an increased copy number may lead to an unfavorable prognosis. Cancer 2007. © 2007 American Cancer Society. [source] Intracrine androgenic apparatus in human bone marrow stromal cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tarvo Sillat Abstract It was suggested that human mesenchymal stromal cells might contain an intracrine enzyme machinery potentially able to synthesize the cell's own supply of dihydrotestosterone (DHT) from dehydroepiandrosterone (DHEA) pro-hormone produced in the adrenal cortex in the reticular zone, which is unique to primates. Indeed, 3,-hydroxysteroid dehydrogenase (3,-HSD) and 5,-reductase enzyme proteins were expressed in resting mesenchymal stromal cells (MSCs) in vitro. However, the ,bridging' enzymes 17,-HSDs, catalysing interconversion between 17,-ketosteroids and 17,-hydroxysteroids, were not found in resting MSCs, but 17,-HSD enzyme protein was induced in a dose-dependent manner by DHEA. Quantitative real-time polymerase chain reactions disclosed that this was mainly due to induction of the isoform 5 catalysing this reaction in ,forward', androgen-bound direction (P < 0.01). This work demonstrates that the MSCs have an intracrine machinery to convert DHEA to DHT if and when challenged by DHEA. DHEA as substrate exerts a positive, feed-forward up-regulation on the 17,-hydroxy steroid dehydrogenase-5, which may imply that DHEA-DHT tailor-making in MSCs is subjected to chronobiological regulation. [source] Plasma renin in mice with one or two renin genesACTA PHYSIOLOGICA, Issue 4 2004P. B. Hansen Abstract Aim:, In the present study we have investigated whether the presence of a second renin gene exerts an overriding influence on plasma renin such that mice with two renin genes have consistently higher renin levels than mice with only one renin gene. Methods:, Plasma renin was determined as the rate of angiotensin I generation using a radioimmunoassay (RIA) kit with (plasma renin concentration, PRC) or without (plasma renin activity, PRA) the addition of purified rat angiotensinogen as substrate. Results:, In male 129SvJ, DBA/2 and Swiss Webster mice, strains possessing both Ren-1 and Ren-2, PRC (ng Ang I mL,1 h,1) averaged 178 ± 36, 563 ± 57 and 550 ± 43 while PRA was 2.9 ± 0.5, 3.6 ± 0.8 and 7.8 ± 1.2. In male C57BL/6, C3H and BALB/c mice that express only Ren-1, PRC averaged 426 ± 133, 917 ± 105 and 315 ± 72, and PRA was 3.4 ± 1.0, 6.9 ± 1.7 and 4.5 ± 1.2. In the two renin gene A1AR,/, mice compared with the one renin gene A1AR+/+, PRC averaged 538 ± 321 and 415 ± 159 while PRA averaged 3.2 ± 1.1 and 4.4 ± 1.4 ng Ang I mL,1 h,1. Aldosterone levels showed no significant differences between one renin (C57BL/6, C3H and BALB/c) and two renin (129SvJ, DBA/2 and Swiss Webster) gene mice. Furthermore, by quantitative real-time polymerase chain reaction (RT-PCR) we found no correlation between the number of renin genes and whole kidney renin mRNA levels from one and two renin gene mice. Conclusion:, Our data show that baseline plasma renin is not systematically higher in mice with two renin genes than in one renin gene mice. Thus, the presence of a second renin gene does not seem to be a major determinant of differences in PRC between different mouse strains. [source] Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2009Sebastian Kreuz Abstract Background Acetyl-CoA carboxylases (ACC) 1 and 2 are central enzymes in lipid metabolism. To further investigate their relevance for the development of obesity and type 2 diabetes, expression of both ACC isoforms was analyzed in obese fa/fa Zucker fatty and Zucker diabetic fatty rats at different ages in comparison to Zucker lean controls. Methods ACC1 and ACC2 transcript levels were measured by quantitative real-time polymerase chain reaction in metabolically relevant tissues of Zucker fatty, Zucker diabetic fatty and Zucker lean control animals. Quantitative real-time polymerase chain reaction was also applied to measure ACC tissue distribution in human tissues. For confirmation on a protein level, quantitative mass spectrometry was used. Results Disease-related transcriptional changes of both ACC isoforms were observed in various tissues of Zucker fatty and Zucker diabetic fatty rats including liver, pancreas and muscle. Changes were most prominent in oxidative tissues of diabetic rats, where ACC2 was significantly increased and ACC1 was reduced compared with Zucker lean control animals. A comparison of the overall tissue distribution of both ACC isoforms in humans and rats surprisingly revealed strong differences. While in rats ACC1 was mainly expressed in lipogenic and ACC2 in oxidative tissues, ACC2 was predominant in oxidative and lipogenic tissues in humans. Conclusion Our data support a potential role for both ACC isoforms in the development of obesity and diabetes in rats. However, the finding of fundamental species differences in ACC1 and ACC2 tissue expression might be indicative for different functions of both isoforms in humans and rats and raises the question to which degree these models are predictive for the physiology and pathophysiology of lipid metabolism in humans. Copyright © 2009 John Wiley & Sons, Ltd. [source] Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practicesENVIRONMENTAL MICROBIOLOGY, Issue 9 2007Ji-zheng He Summary The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated by using quantitative real-time polymerase chain reaction, cloning and sequencing approaches based on amoA genes. The soil, classified as agri-udic ferrosols with pH (H2O) ranging from 3.7 to 6.0, was sampled in summer and winter from long-term field experimental plots which had received 16 years continuous fertilization treatments, including fallow (CK0), control without fertilizers (CK) and those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK, PK, NPK and NPK plus organic manure (OM). Population sizes of AOB and AOA changed greatly in response to the different fertilization treatments. The NPK + OM treatment had the highest copy numbers of AOB and AOA amoA genes among the treatments that received mineral fertilizers, whereas the lowest copy numbers were recorded in the N treatment. Ammonia-oxidizing archaea were more abundant than AOB in all the corresponding treatments, with AOA to AOB ratios ranging from 1.02 to 12.36. Significant positive correlations were observed among the population sizes of AOB and AOA, soil pH and potential nitrification rates, indicating that both AOB and AOA played an important role in ammonia oxidation in the soil. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from different treatments were affiliated with Nitrosospira or Nitrosospira- like species and grouped into cluster 3, and little difference in AOB community composition was recorded among different treatments. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). Cluster M dominated exclusively in the N, NP, NK and PK treatments, indicating a pronounced difference in the community composition of AOA in response to the long-term fertilization treatments. These findings could be fundamental to improve our understanding of the importance of both AOB and AOA in the cycling of nitrogen and other nutrients in terrestrial ecosystems. [source] Long-term effects of idiotype vaccination on the specific T-cell response in peripheral blood and bone marrow of multiple myeloma patientsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2007Amir Osman Abdalla Abstract Objectives:, To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). Materials and methods:, Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (,-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. Results:, At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th2 cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) (P < 0.02) and shifted at the gene level from a Th1 to a Th2 profile (P < 0.05). Conclusion:, Id-specific T cells may decline overtime and shift toward a Th2 response and may be found at a similar frequency of patients in blood and BM. [source] Changes in alternative brain-derived neurotrophic factor transcript expression in the developing human prefrontal cortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2009Jenny Wong Abstract In this study, we determined when and through which promoter brain-derived neurotrophic factor (BDNF) transcription is regulated during the protracted period of human frontal cortex development. Using quantitative real-time polymerase chain reaction, we examined the expression of the four most abundant alternative 5, exons of the BDNF gene (exons I, II, IV, and VI) in RNA extracted from the prefrontal cortex. We found that expression of transcripts I,IX and VI,IX was highest during infancy, whereas that of transcript II,IX was lowest just after birth, slowly increasing to reach a peak in toddlers. Transcript IV,IX was significantly upregulated within the first year of life, and was maintained at this level until school age. Quantification of BDNF protein revealed that levels followed a similar developmental pattern as transcript IV,IX. In situ hybridization of mRNA in cortical sections showed the highest expression in layers V and VI for all four BDNF transcripts, whereas moderate expression was observed in layers II and III. Interestingly, although low expression of BDNF was observed in cortical layer IV, this BDNF mRNA low-zone decreased in prominence with age and showed an increase in neuronal mRNA localization. In summary, our findings show that dynamic regulation of BDNF expression occurs through differential use of alternative promoters during the development of the human prefrontal cortex, particularly in the younger age groups, when the prefrontal cortex is more plastic. [source] Trefoil factor family 3 expression in the oral cavityEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 6 2009T. Storesund This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3-positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity. [source] Amplification and overexpression of prosaposin in prostate cancerGENES, CHROMOSOMES AND CANCER, Issue 4 2005Shahriar Koochekpour We identified prosaposin (PSAP) as a secreted protein expressed in androgen-independent (AI) prostate cancer cells by cloning/sequencing, after probing a PC-3 cDNA library expressed in the ,TriplEx phagemid expression vector with a polyclonal rabbit antibody generated against pooled human seminal plasma. PSAP is a neurotrophic molecule; its deficiency or inactivation has proved to be lethal in man and mice, and in mice, it leads to abnormal development and atrophy of the prostate gland, despite normal testosterone levels. We used Southern hybridization, quantitative real-time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis, and we now report the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC-3, DU-145, MDA-PCa 2b, M-12, and NCI-H660. In addition, by using SNP arrays and a set of 25 punch biopsy samples of human prostate cancer xenografts (LAPC3, LuCaP 23.1, 35, 49, 58, 73, 77, 81, 86.2, 92.1, 93, 96, 105, and 115), lymph nodes, and visceral-organ metastases, we detected amplification of the PSAP locus (10q22.1) in LuCaP 58 and 96 xenografts and two lymph node metastases. In addition, AI metastatic prostate cancer cell lines C4-2B and IA8-ARCaP over-expressed PSAP mRNA without evidence of genomic amplification. Taken together with prior data that demonstrated the growth-, migration-, and invasion-promoting activities, the activation of multiple signal transduction pathways, and the antiapoptotic effect of PSAP (or one of its active domains, saposin C) in prostate cancer cells, our current observation of PSAP amplification or overexpression in prostate cancer suggests, for the first time, a role for this molecule in the process of carcinogenesis or cancer progression in the prostate. © 2005 Wiley-Liss, Inc. [source] The Helicobacter hepaticus hefA Gene is Involved in Resistance to AmoxicillinHELICOBACTER, Issue 1 2009Clara Belzer Abstract Background:, Gastrointestinal infections with pathogenic Helicobacter species are commonly treated with combination therapies, which often include amoxicillin. Although this treatment is effective for eradication of Helicobacter pylori, the few existing reports are less clear about antibiotic susceptibility of other Helicobacter species. In this study we have determined the susceptibility of gastric and enterohepatic Helicobacter species to amoxicillin, and have investigated the mechanism of amoxicillin resistance in Helicobacter hepaticus. Materials and methods:, The minimal inhibitory concentration (MIC) of antimicrobial compounds was determined by E -test and agar/broth dilution assays. The hefA gene of H. hepaticus was inactivated by insertion of a chloramphenicol resistance gene. Transcription was measured by quantitative real-time polymerase chain reaction. Results:, Three gastric Helicobacter species (H. pylori, H. mustelae, and H. acinonychis) were susceptible to amoxicillin (MIC < 0.25 mg/L). In contrast, three enterohepatic Helicobacter species (H. rappini, H. bilis, and H. hepaticus) were resistant to amoxicillin (MIC of 8, 16, and 6,64 mg/L, respectively). There was no detectable ,-lactamase activity in H. hepaticus, and inhibition of ,-lactamases did not change the MIC of amoxicillin of H. hepaticus. A H. hepaticus hefA (hh0224) mutant, encoding a TolC-component of a putative efflux system, resulted in loss of amoxicillin resistance (MIC 0.25 mg/L), and also resulted in increased sensitivity to bile acids. Finally, transcription of the hefA gene was not responsive to amoxicillin, but induced by bile acids. Conclusions:, Rodents are frequently colonized by a variety of enterohepatic Helicobacter species, and this may affect their global health status and intestinal inflammatory responses. Animal facilities should have treatment strategies for Helicobacter infections, and hence resistance of enterohepatic Helicobacter species to amoxicillin should be considered when designing eradication programs. [source] Naturally Occurring Regulatory T cells (CD4+, CD25high, FOXP3+) in the Antrum and Cardia are Associated with Higher H. pylori Colonization and Increased Gene Expression of TGF-,1HELICOBACTER, Issue 4 2008Arne Kandulski Abstract Background:Helicobacter pylori causes gastric inflammation. Despite the induction of H. pylori -specific B- and T cells, the immune response is not sufficient to clear the infection. Regulatory T cells (Treg cells) suppress the activation and proliferation of antigen-specific T cells and mediate immunologic tolerance. FOXP3 was shown to be expressed in a subset of Treg cells known as ,naturally occurring Treg cells'. These cells have not been sufficiently studied in context to H. pylori -induced inflammation in human gastric mucosa. Materials and methods: The study included 76 patients stratified according to the presence of H. pylori. Gene expression levels of FOXP3, transforming growth factor (TGF)-,1, and interleukin-10 were analyzed by quantitative real-time polymerase chain reaction in biopsies from gastric antrum, corpus, and cardia. FOXP3 expression was also analyzed by immunohistochemistry. Differences in expression levels were analyzed by comprehensive statistical analyses and correlated with clinical and histomorphologic parameters. Results:H. pylori -positive patients revealed a 19- to 25-fold induction of FOXP3 transcript levels in antrum and cardia (p < .02). FOXP3 transcript levels correlated positively with inflammation (p < .04) and TGF-,1 transcript levels (p < .001). Furthermore, a positive correlation between FOXP3+ Treg cells and H. pylori colonization was demonstrated. Conclusion: This study demonstrates that H. pylori -induced gastritis is associated with a recruitment of naturally occurring FOXP3+ Treg cells that correlates with the degree of bacterial colonization and mucosal TGF-,1 expression. Together, these data support the hypothesis that naturally FOXP3+ Treg cells play a role in the lifelong persistence of H. pylori infection in humans. [source] Helicobacter pylori Stimulates a Mixed Adaptive Immune Response with a Strong T-Regulatory Component in Human Gastric MucosaHELICOBACTER, Issue 3 2007Rasmus Goll Abstract Background:, Host factors play an important role in the pathophysiology of Helicobacter pylori infection and development of gastritis and related disease. The established opinion is that the T-cell-mediated immune response to H. pylori infection is of Th1 type. Our earlier immune cell phenotype studies indicate a mixed Th1,Th2 profile of the effector cells. Therefore, an extensive adaptive and regulatory cytokine gene expression profile was conducted by quantitative real-time polymerase chain reaction (qPCR). Materials and Methods:, Biopsies from gastric mucosa of 91 patients diagnosed as H. pylori negative, H. pylori positive with gastritis, or H. pylori positive with peptic ulcer were obtained by endoscopy. Gene expressions of nine cytokines and CagA status were measured by qPCR. Results:, All cytokine genes showed higher expression levels in the presence of H. pylori when compared to H. pylori- negative samples (fold increase: IL8: × 11.2; IL12A: × 2.4; TNF-,: × 5.2; IFN-,: × 4.3; IL4: × 3.6; IL6: × 14.7; and IL10: × 6.7). Patients infected with CagA-positive strains had higher expression of IL1-, and IL18 compared to patients infected with CagA-negative strains (× 1.6 for IL1-, and × 2.0 for IL18). Patients with duodenal ulcer had a lower antral Th1/Th2 ratio than other H. pylori -positive patients. Conclusions:, The cytokine profile of H. pylori -infected gastric mucosa shows a mixed Th1,Th2 profile. Furthermore, a high IL10 expression may indicate that also regulatory T cells play a role in the chronic phase of H. pylori infection. [source] Therapy adapted to molecular response in patients with chronic myelogenous leukaemia in first chronic phase: results of the Duesseldorf study,HEMATOLOGICAL ONCOLOGY, Issue 4 2008Frank Neumann Abstract This study evaluates response-adapted treatment of chronic myelogenous leukaemia (CML) in chronic phase using molecular response criteria. bcr-abl/G6PDH ratios were assessed by Light-Cycler quantitative real-time polymerase chain reaction (PCR( in 277 peripheral blood samples from 33 patients, before and every 3 months during therapy. Sixty-six per cent (22/33) of the patients fulfiled our molecular response criterion of ,1 log decrease in bcr-abl transcript after 6 or ,2 log decrease after 9 and every following 3 months. Dose escalation was necessary for 33% (11/33) of the patients. Of these, 54% (6/11) achieved a reduction of bcr-abl mRNA by ,2 log (n,=,3) or ,3 log (n,=,3) with 800,mg Imatinib. Forty-five per cent (5/11) showed insufficient molecular response with 800,mg Imatinib and received Nilotinib. In conclusion, the assessment of molecular response permits an individual patient-tailored treatment of CML in first chronic phase, resulting in the majority of patients achieving a major molecular response after 2 years of therapy. Copyright © 2008 John Wiley & Sons, Ltd. [source] UbcH10 expression in human lymphomasHISTOPATHOLOGY, Issue 6 2009Giancarlo Troncone Aims:, The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. Methods and results:, Cell lines and tissue samples of Hodgkin's lymphoma (HL) and of non-Hodgkin's lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt's lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G2/M phase and dramatically decreasing in G0/G1 phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. Conclusions:, Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting. [source] Preserved Na+/H+ exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis,INFLAMMATORY BOWEL DISEASES, Issue 7 2010Sunil Yeruva PhD Abstract Background: A major causative factor of diarrhea in ulcerative colitis (UC) patients is the loss of Na+ absorptive capacity of the inflamed colonic mucosa. Potential contributing mechanisms include reduced driving force for active transport, and impaired expression, mislocalization, or defective transport function of Na+ absorptive proteins. We therefore studied the expression, brush border membrane (BBM) localization, and transport capacity of the major intestinal Na+ absorptive protein, the Na+/H+ exchanger isoform 3 (NHE3) in biopsies from UC patients. Methods: In UC and control biopsies, inflammation was graded histologically, NHE3, tumor necrosis factor alpha (TNF-,), villin, as well as other housekeeping genes were analyzed by quantitative real-time polymerase chain reaction (PCR), BBM localization of NHE3 determined by immunohistochemistry, and confocal microscopy. Na+ absorptive capacity was assessed by 22Na+ isotope fluxes and NHE3 transport activity measured microfluorometrically in BCECF-loaded surface colonocytes within isolated crypts. Results: In mildly, moderately, and severely inflamed sigmoid colon of UC patients, neither NHE3 mRNA expression nor the abundance of NHE3 in the BBM was significantly altered compared to other structural components of the BBM. However, Na+ absorption was strongly reduced by ,80% and acid-activated NHE3 transport activity was significantly decreased in the surface cells of sigmoid colonic crypts even in moderately inflamed mucosa. Conclusions: In the colonic mucosa of patients with active UC, NHE3 transport capacity was found significantly decreased despite correct NHE3 location and abundance in the brush border, independent of current treatment. These findings suggest functional NHE3 transport as a novel factor for inflammatory diarrhea in UC patients. (Inflamm Bowel Dis 2010) [source] Low counts of Faecalibacterium prausnitzii in colitis microbiotaINFLAMMATORY BOWEL DISEASES, Issue 8 2009H. Sokol MD Abstract Background: The intestinal microbiota is suspected to play a role in colitis and particularly in inflammatory bowel disease (IBD) pathogenesis. The aim was to compare the fecal microbiota composition of patients with colitis to that of healthy subjects (HS). Methods: fecal samples from 22 active Crohn's disease (A-CD) patients, 10 CD patients in remission (R-CD), 13 active ulcerative colitis (A-UC) patients, 4 UC patients in remission (R-UC), 8 infectious colitis (IC) patients, and 27 HS were analyzed by quantitative real-time polymerase chain reaction (PCR) targeting the 16S rRNA gene. Bacterial counts were transformed to logarithms (Log10 CFU) for statistical analysis. Results: Bacteria of the phylum Firmicutes (Clostridium leptum and Clostridium coccoides groups) were less represented in A-IBD patients (9.7; P = 0.004) and IC (9.4; P = 0.02), compared to HS (10.8). Faecalibacterium prausnitzii species (a major representative of the C. leptum group) had lower counts in A-IBD and IC patients compared to HS (8.8 and 8.3 versus 10.4; P = 0.0004 and P = 0.003). The Firmicutes/Bacteroidetes ratio was lower in A-IBD (1.3; P = 0.0001) and IC patients (0.4; P = 0.002). Compared to HS, Bifidobacteria were less represented in A-IBD and IC (7.9 and 7.7 versus 9.2; P = 0.001 and P = 0.01). Conclusions: The fecal microbiota of patients with IBD differs from that of HS. The phylum Firmicutes and particularly the species F. prausnitzii, are underrepresented in A-IBD patients as well as in IC patients. These bacteria could be crucial to gut homeostasis since lower counts of F. prausnitzii are consistently associated with a reduced protection of the gut mucosa. (Inflamm Bowel Dis 2009) [source] Enhanced expression of MMP-7 and MMP-13 in inflammatory bowel disease: A precancerous potential?INFLAMMATORY BOWEL DISEASES, Issue 11 2006Dr. Timo Rath PhD Abstract Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP-2, MMP-9, MMP-7, and MMP-13. The aim of this study was to determine the expression levels of MMP-2, MMP-9, MMP-7, MMP-13, and MMP-14 and their specific inhibitor TIMP-1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohn's disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohn's disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real-time polymerase chain reaction to study MMP and TIMP-1 gene expression in both pathological and normal mucosal specimens. For MMP-2, MMP-9, and TIMP-1, protein expression also was quantified with sandwich enzyme-linked immunosorbent assay. In biopsy specimens of Crohn's disease and ulcerative colitis, significantly increased levels of MMP-2, MMP-7, and MMP-13 mRNA were found. MMP-2 and MMP-9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP-7 and MMP-13 genes. MMP-14 mRNA was decreased in APs. MMPs, especially MMP-7 and MMP-13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP-7 and MMP-13 may serve as an early indicator for colorectal carcinogenesis. [source] Spatial distribution and differential expression of the PBAN receptor in tissues of adult Helicoverpa spp. (Lepidoptera: Noctuidae)INSECT MOLECULAR BIOLOGY, Issue 3 2007A. Rafaeli Abstract Pheromone-biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone production in many female moths. PBAN-like peptides, with common FXPRLamide C-terminals are found in other insect groups where they have other functions. The ubiquity and multifunctional nature of the pyrokinin/PBAN family of peptides suggests that the PBAN receptor proteins could also be present in a variety of insect tissues with alternative functions from that of sex pheromone biosynthesis. Previously we showed the presence of the PBAN-R in Helicoverpa armigera at the protein level. In the present study we confirm the similarities between the two Helicoverpa species: armigera and zea by (1) demonstrating the presence of the receptor protein in Sf9 cells, cloned to express the HezPBAN receptor, as compared with the endogenous receptor protein, previously shown in H. armigera pheromone glands, and (2) by identifying the nucleotide sequence of the PBAN-R from mRNA of H. armigera pheromone glands. Sequences of the two Helicoverpa spp. are 98% identical with most changes taking place in the 3,-end. We demonstrate the spatial distribution of the PBAN receptor protein in membranes of H. armigera brain (Br), thoracic ganglion (TG) and ventral nerve cord (VNC). We also demonstrate the presence and differential expression of the PBAN receptor gene (using reverse transcription,polymerase chain reaction and reverse transcription,quantitative real-time polymerase chain reaction, respectively) in the neural tissues (Br, TG and VNC) of adult H. armigera female moths as compared with its presence in pheromone glands. Surprisingly, the gene for the PBAN receptor is also detected in the male tissue homologous to the female pheromone gland, the aedeagus, although the protein is undetectable and PBAN does not induce physiological (pheromone production) or cellular (cyclic-adenosine monophosphate production) responses in this tissue. Our findings indicate that PBAN or PBAN-like receptors are present in the neural tissues and may represent a neurotransmitter-like function for PBAN-like peptides. In addition, the surprising discovery of the presence of the gene encoding the PBAN receptor in the male homologous tissue, but its absence at the protein level, launches opportunities for studying molecular regulation pathways and the evolution of these G protein coupled receptors (GPCRs). [source] | |||||||||||||||||||||||||||||||||||||