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DPI
Selected AbstractsGlial cell loss, proliferation and replacement in the contused murine spinal cordEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Judith M. Lytle Abstract Studies in the rat have shown that contusive spinal cord injury (SCI) results in devastating pathology, including significant loss of mature oligodendrocytes and astrocytes even in spared white matter. Subsequently, there is increased proliferation of endogenous NG2+ cells, postulated to contribute to replacement of mature glia chronically, which is important for functional recovery. Studies of mechanisms that stimulate endogenous progenitor cells would be facilitated by using mouse models with naturally occurring and genetically engineered mutations. To determine whether the murine response is similar to that in the rat, we performed contusive SCI on adult female C57Bl/6 mice at the T8,9 level. Animals received bromodeoxyuridine injections in the first week following injury and were killed at 1, 3, 4, 7 or 28 days postinjury (DPI). The overall loss of macroglia and the temporal,spatial response of NG2+ cells after SCI in the (C57Bl/6) mouse was very similar to that in the (Sprague,Dawley) rat. By 24 h after SCI nearly half of the macroglia in spared ventral white matter had been lost. Cell proliferation was increased at 1,7 DPI, peaking at 3,4 DPI. Dividing cells included NG2+ cells and Cd11b+ macrophages and microglia. Furthermore, cells dividing in the first week expressed markers of mature glia at 28 DPI. The similarities in endogenous progenitor cell response to SCI in the mouse and rat suggest that this is a fundamental injury response, and that transgenic mouse models may be used to further probe how this cellular response to SCI might be enhanced to improve recovery after SCI. [source] Gangliosides activate microglia via protein kinase C and NADPH oxidaseGLIA, Issue 3 2004Kyoung-Jin Min Abstract Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)-1,, tumor necrosis factor-, (TNF-,), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL-1,, TNF-,, and iNOS in ganglioside-treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Gö6976, Ro31-8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC-,, ,II, and , and NADPH oxidase p67phox translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside-induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside-induced activation of NF-,B, an essential transcription factor that mediates the expression of IL-1,, TNF-,, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF-,B. © 2004 Wiley-Liss, Inc. [source] Chronic ethanol increases adeno-associated viral transgene expression in rat liver via oxidant and NF,B-dependent mechanismsHEPATOLOGY, Issue 5 2000Michael D. Wheeler Recombinant adeno-associated virus (rAAV) transduction is limited in vivo, yet can be enhanced by hydroxyurea, ultraviolet-irradiation, or adenovirus coinfection, possibly via mechanisms involving stress in the host cell. Because chronic ethanol induces oxidative stress, it was hypothesized that chronic ethanol would increase rAAV transduction in vivo. To test this hypothesis, rAAV encoding ,-galactosidase was given to Wistar rats that later received either ethanol diet or high-fat control diet via an enteral-feeding protocol for 3 weeks. Expression and activity of ,-galactosidase in the liver were increased nearly 5-fold by ethanol. The increase in transgene expression was inhibited by antioxidant diphenylene iodonium (DPI), which is consistent with the hypothesis that ethanol causes an increase in rAAV transduction via oxidative stress. Ethanol increased DNA synthesis only slightly; however, it increased the nuclear transcription factor ,B (NF,B) 4-fold, a phenomenon also sensitive to DPI. Moreover, a 6-fold increase in rAAV transgene expression was observed in an acute ischemia-reperfusion model of oxidative stress. Transgene expression was transiently increased 24 hours after ischemia-reperfusion 3 days and 3 weeks after rAAV infection. Further, adenoviral expression of superoxide dismutase or I,B, superrepressor inhibited rAAV transgene expression caused by ischemia-reperfusion. Therefore, it is concluded that ethanol increases rAAV transgene expression via mechanisms dependent on oxidative stress, and NF,B likely through enhancement of cytomegaloviral (CMV) promoter elements. Alcoholic liver disease is an attractive target for gene therapy because consumption of ethanol could theoretically increase expression of therapeutic genes (e.g., superoxide dismutase). Moreover, this study has important implications for rAAV gene therapy and potential enhancement and regulation of transgene expression in liver. [source] Effects of Isoproterenol and Amiodarone on the Double Potential Interval After Ablation of the Cavotricuspid IsthmusJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 9 2003HIROSHI TADA M.D. Introduction: A corridor of double potentials along the ablation line has been recognized to be an indicator of complete cavotricuspid isthmus block. Isoproterenol is used to confirm cavotricuspid isthmus block, but the effects of isoproterenol on the double potential interval (DPI), either in the absence or presence of amiodarone, are unknown. Methods and Results: Thirty-two patients with isthmus-dependent atrial flutter underwent successful ablation of the cavotricuspid isthmus. The procedure was performed in the drug-free state in 23 patients, and 2 to 7 days after discontinuation of chronic amiodarone therapy in 9 patients. Electrograms recorded along the ablation line before and during isoproterenol infusion were analyzed after isthmus block was achieved. Double potentials were recorded along the entire ablation line upon achievement of complete isthmus block in all patients. The DPI in 9 patients treated with amiodarone was longer than in the other patients (147 ± 32 msec vs 119 ± 19 msec, P < 0.001). The DPI increased as the pacing cycle length shortened in patients treated with amiodarone, but not in the other patients. At all pacing cycle lengths, isoproterenol shortened the DPI to a greater extent in the patients treated with amiodarone than in the other patients. Conclusion: Amiodarone results in rate-dependent prolongation of the DPI during coronary sinus pacing after ablation of the cavotricuspid isthmus. Isoproterenol shortens the DPI despite the presence of complete isthmus block, and this effect is accentuated in the presence of amiodarone. (J Cardiovasc Electrophysiol, Vol. 14, pp. 935-939, September 2003) [source] iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2008Feng Wu Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-, (LPS,+,IFN,)-stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS,+,IFN, caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS,+,IFN, induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NF,B activation. LPS,+,IFN, stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of I,B, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS,+,IFN,-induced DNA binding activity of transcription factors IRF1 and AP1 but not NF,B. In conclusion, LPS,+,IFN,-induced NF,B activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS,+,IFN,-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NF,B activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis. J. Cell. Physiol. 217: 207,214, 2008. © 2008 Wiley-Liss, Inc. [source] Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: Role of p42/44 mitogen-activated protein kinase and reactive oxygen species,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2001Zhonglin Xie Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen-activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 ,M, 16 h) increased osteopontin expression (fold increase 3.3±0.34, n,=,12, P,<,0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time- (within 4 h) and concentration-dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho-specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II-stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40,60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley-Liss, Inc. [source] NADPH oxidase inhibitor diphenyliodonium abolishes lipopolysaccharide-induced down-regulation of transferrin receptor expression in N2a and BV-2 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2006Katarina Reis Abstract The activation of cellular inflammatory response is tightly linked to induced production of reactive oxygen species (ROS) and nitric oxide (NO), which in turn have been identified as important regulators of cellular iron metabolism. In the present study, we have used the microglia cell line BV-2 and the neuroblastoma cell line N2a to study the regulatory effects of the microbial agent lipopolysaccharide (LPS) on the expression of the transferrin receptor (TfR) and ferritin in cell lines with different characteristics. The receptor mainly responsible for LPS recognition is the Toll-like receptor 4 (TLR4) that triggers a variety of intracellular signalling cascades leading to the induction of transcription of target genes involved in the innate immune response. Among the pathways to be activated is the MAPK cascade leading to the activation of nuclear factor-,B that induces transcription of a variety of genes, e.g., inducible nitric oxide synthase (iNOS). The TLR4-mediated LPS response also induces the production of ROS through a mechanism(s) suggested to involve the activation of NADPH oxidase(s). This study shows that exposure of BV-2 and N2a cells to LPS results in decreased TfR protein levels and increased H-ferritin mRNA levels. The LPS down-regulatory effect on TfR protein expression is abolished by the NADPH oxidase inhibitor diphenyliodonium (DPI) but is not affected by the free radical scavenger N-acetyl-L-cysteine (NAC) or the iNOS inhibitor aminoguanidine (AG). The increased H-ferritin mRNA levels in response to LPS are not affected by DPI, NAC, or AG. © 2006 Wiley-Liss, Inc. [source] Cospray dried antibiotics for dry powder lung deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2008Handoko Adi Abstract The aim of this study was to assess the potential of delivering a combination antibiotic therapy, containing doxycycline and ciprofloxacin (both hydrochloride) as a dry powder (DPI) formulation for inhalation. Single and combination antibiotics were produced by spray drying. Particle size distributions were characterized by laser diffraction and imaging conducted by scanning electron microscopy. Solid-state characterisation of the antibiotics was carried out using differential scanning calorimetry, dynamic vapour sorption, X-ray powder diffraction, and differential scanning calorimetry. Using the Aerolizer® device, the aerosol performance was measured using multistage liquid impinger and analysed using high performance liquid chromatography (R2,=,1.0, CV,=,0.4,1.0%). Furthermore, a disk diffusion test was performed for the assessment of the in vitro antimicrobial activity of the raw and spray dried antibiotics against bacteria. Results showed that cospray drying of the ciprofloxacin and doxycycline produced an antibiotic formulation (in a 1:1 ratio) suitable for inhalation that showed to be physically more stable then the analogous single spray dried antibiotic. The cospray dried powder has improved dispersion over the less stable single spray dried ciprofloxacin. The spray dried antibiotics were observed to have similar antimicrobial activity to the original antibiotics for Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyrogenes, suggesting the spray drying process does not affect the anti-bacterial activity of the drug. Cospray dried antibiotics from a DPI is thus feasible and can potentially be an attractive delivery alternative to the more conventional systemic delivery route. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 3356,3366, 2008 [source] Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinoneJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2005Yaxian Ding NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity. [source] ETHANOL-INDUCED SUPEROXIDE RADICALS IN FETAL CORTICAL NEURONS: CELLULAR ROS NETWORKALCOHOLISM, Issue 2008Amina E Jamali Alcohol exposure to the developing brain compromises both neurons and glial functions. While neurons are considered the primary targets, microglia may play a neurotoxic role in this process. Previous studies demonstrated that neuron death is due to oxidative stress and mitochondrially mediated (Intrinsic). These studies showed a rapid increase (within minutes) in reactive oxygen species (ROS). Due to the diffusive nature of ethanol and multiple sources of free radicals, we sought to determine the primary source of superoxide targeted by ethanol. Confocal studies of neurons suggest that the superoxide radicals may originate from the mitochondria. Using whole neurons in a luminol-based chemiluminescence assay (Diogenes) we detected superoxide radicals in the extracellular mileu. We observed a two-three fold transient increase in the steady state generation of superoxide radicals between 20 minutes to one hour of ethanol exposure (4mg/ml). However, the presence of Rotenone (mitochondrial complex I inhibitor) and DPI (an inhibitor of all flavinoids) blocked the release of these superoxide radicals. Interestingly, cortical microglia treated identically with ethanol, showed a greater than five fold increase in superoxide generation with a maximum at one hour. Moreover, since ethanol is known to induce hydrogen peroxide generation, it was used as a mimetic. Hydrogen peroxide also induced the production of superoxide different time kinetics. Thus, together these data demonstrate that ethanol induces the steady state production of superoxide radicals in the extracellular mileu in a mitochondrial dependent manner. Since NOX2 an NADPH oxidase is expressed in neurons, it is a potential candidate for the secondary sites of superoxide generation. The ROS network between mitochondria and the plasma membrane highlights new therapeutical targets to counter ethanol toxicity. [source] Majority of children aged 3 years and above can reliably inhale through the ClickhalerPEDIATRIC PULMONOLOGY, Issue 1 2003Shaique M. Iqbal MRCPCH Abstract Guidelines suggest that pressurized metered dose inhalers (pMDI) plus spacers are the delivery system of choice for children. However, they are bulky, which makes them inconvenient. It was suggested that the smaller dry-powder inhalers (DPIs) may be suitable for delivering short-acting bronchodilators to preschool children. This study considered whether preschool children could reliably generate sufficient inspiratory flows to use the Clickhaler DPI. Twenty-seven asthmatic and 34 nonasthmatic children, aged 2,5 years, were recruited. Following training, they were asked to inhale four times through a Clickhaler flow monitoring system, twice "steadily" and twice "forcefully." Inspiratory flow data were collected during each inhalation. Of the 3-, 4-, and 5-year-old asthmatics, 62.5, 100, and 100%, respectively, could reliably differentiate between inhaling and exhaling through the DPI. For nonasthmatics, the figures were 66, 60, and 88%, respectively. All but one of the children who understood the instructions generated a PIF of greater than 15 l/min (range, 13.9,88.3 l/min and 21.2,84.5 l/min in asthmatic and nonasthmatic children, respectively). These data indicate that the majority of children aged 3 years and above could reliably inhale rather than exhale through a dry-powder inhaler, and that they generate inspiratory flows sufficient to use the Clickhaler effectively. The results indicate that the device could be a suitable delivery system for ,2 -agonists in preschool children. Pediatr Pulmonol. 2003; 36:63,68. © 2003 Wiley-Liss, Inc. [source] Sensitivity of Turbutester and Accuhaler tester in asthmatic children and adolescentsPEDIATRICS INTERNATIONAL, Issue 1 2010Wiparat Manuyakorn Abstract Background:, Dry powder inhalers (DPI) are alternative devices for delivering medication for treatment of asthma. The amount of drug delivery to the lungs is directly influenced by peak inspiratory flow rate (PIFR). A minimum PIFR of ,30 L/min is needed for the Turbuhaler and Accuhaler. Methods:, In order to evaluate the sensitivity of the Turbutester and Accuhaler tester in detecting the minimum and optimum PIFR for the Turbuhaler and Accuhaler in asthmatic children, PIFR was measured using the In-Check Dial through the internal resistance of the Turbuhaler and Accuhaler and compared according to the child's ability to make a whistle sound via both testers. Results:, A total of 259 asthmatic children were studied: 20 pre-school children, aged 5,6 years; 174 school-age children, aged 7,12 years; and 65 adolescents, aged 13,18 years. The sensitivity of the Turbutester and Accuhaler tester to detect optimum PIFR were 98.40% and 97.2%, respectively. In the comparison among age groups, the sensitivity of the Accuhaler tester to detect optimum or minimum PIFR for the Accuhaler was 95%, 97.7% and 95.4%, respectively. The sensitivity of the Turbutester to detect optimum PIFR for the Turbuhaler was 94.4%, 98.8% and 98.5%, respectively. The sensitivity of the Turbutester to detect minimum PIFR for the Turbuhaler was 94.7%, 100% and 100%, respectively. There were no significant differences in percentage of having optimum or minimum PIFR among asthma severity and current device usage in all age groups. Conclusions:, Most children aged at least 5 years could generate enough PIFR to use dry powder inhaler devices. Both the Turbutester and Accuhaler tester were found to have high sensitivity in detecting optimum and minimum required PIFR. [source] Ability of preschool children to use dry powder inhalers as evaluated by In-Check MeterPEDIATRICS INTERNATIONAL, Issue 1 2006YOKO S ADACHI Abstract Background: Although current guidelines recommend the pressurized metered-dose inhaler with a spacer for preschool children with asthma, dry powder inhalers (DPI) may be a valuable treatment alternative. Methods: To evaluate the ability of preschool children to inhale through DPI, peak inspiratory flow rates (PIFR) of 57 healthy children aged 3,6 years were measured with In-Check Meter after practising with an instructor. Two different calibrated resistances were attached to the Meter to mimic the internal resistance of each inhaler; Diskus and Turbuhaler. Results: The ability of children to generate adequate inspiratory flow increased with age. The percentages of the 3-, 4-, 5-, and 6-year-old children who were able to inhale reliably through the devices were 30% (3/10), 79.0% (15/19), 100% (16/16), and 100% (12/12), respectively. In these children, 100%, 93.3%, 100%, and 100% achieved an adequate PIFR for the Diskus (30 L/min). In contrast, 66.7%, 66.7%, 62.5%, and 91.7% generated an adequate PIFR for the Turbuhaler (60 L/min). Conclusions: The In-Check Meter is a useful device to assess the ability of preschool children to generate adequate PIFR for each inhaler. Most children aged ,5 years could use DPI. [source] Overexpression of rice isoflavone reductase-like gene (OsIRL) confers tolerance to reactive oxygen speciesPHYSIOLOGIA PLANTARUM, Issue 1 2010Sang Gon Kim Isoflavone reductase is an enzyme involved in isoflavonoid biosynthesis in plants. However, rice isoflavone reductase-like gene (OsIRL, accession no. AY071920) has not been unraveled so far. Here, we have characterized its behavior in response to oxidizing agents. Using Northern and Western blot analyses, the OsIRL gene and protein were shown to be down-regulated in young seedling roots treated with reduced glutathione (GSH) and diphenyleneiodonium (DPI), known quenchers of reactive oxygen species (ROS). The OsIRL transcript level in rice suspension-cultured cells was also found to be induced by oxidants such as hydrogen peroxide (H2O2), ferric chloride (FeCl3), methyl viologen (MV) and glucose/glucose oxidase (G/GO), but down-regulated when co-treated with GSH. Furthermore, to investigate whether overexpression of OsIRL in transgenic rice plants promotes resistance to ROS, we generated transgenic rice lines overexpressing the OsIRL gene under an abscisic acid (ABA) inducible promoter. Results showed that the OsIRL transgenic rice line activated by ABA treatment was tolerant against MV and G/GO-induced stress in rice leave and suspension-cultured cells. Our results strongly suggest the involvement of OsIRL in homeostasis of ROS. [source] Laryngeal Findings in Users of Combination Corticosteroid and Bronchodilator Therapy,THE LARYNGOSCOPE, Issue 9 2004Natasha Mirza MD Educational Objective: At the conclusion of this article, the readers should be able to 1) describe the laryngeal findings in patients who use combination therapy for asthma, 2) discuss the mechanism of laryngeal irritation from the use of inhalers, and 3) describe possible mechanisms for reducing laryngeal irritation and secondary dysphonia from the use of inhalers. Objectives: To describe voice changes and laryngeal findings in patients who are started on combination corticosteroid and bronchodilator therapy in the form of a dry powder inhaler (DPI). Study Design: Retrospective, single-subject design. Methods: Retrospective review of 10 consecutive patients meeting inclusion criteria, who presented at the voice center with more than 4 weeks of dysphonia after being started on a combination form of asthma medication for control and maintenance therapy. All patients were nonsmokers and without history of previous identification or excision of vocal pathology. All patients were treated previously with a proton pump inhibitor for gastroesophageal reflux. Laryngeal videostroboscopic evaluations were performed on all patients. Patients were asked to complete a questionnaire regarding their perceived voice change and history of medical maintenance therapy for asthma. Results: Dysphonia was present in the patients selected for greater than 4 weeks. Patients had been switched to combination therapy after previously using traditional two-drug asthma regimens. In eight of nine patients, the vocal folds demonstrated areas of hyperemia, with plaque-like changes on the surface mucosa. Reduced amplitude of vibration and a reduction in mucosal wave propagation were present on videostroboscopy. Questionnaires revealed that all patients were initiated on combination DPI treatment within the last 6 months. Conclusions: Dysphonia caused by a change in the surface mucosa is a side effect from the use of DPI therapy for asthma. The high-impact force during inhalation of the medication and carrier leads to deposition of particles in the upper airway. We believe the extent of mucosal irritation can be minimized by patient education in the proper delivery of DPI. In some cases, however, return of the two medications delivered separately was necessary. The irritation of the laryngeal mucosa and return of normal vibratory parameters occurred in all patients. [source] Nox-2 Is a Modulator of Fibrogenesis in Kidney AllograftsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2009A. Djamali We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with ,-SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (,-SMA+) expressed Nox-2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox-2,. These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and ,-SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (,-SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-,1 and angiotensin II increased Nox-2 mRNA levels 2- to 7-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts. [source] A genetic algorithm for the identification of conformationally invariant regions in protein moleculesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Thomas R. Schneider Understanding macromolecular function often relies on the comparison of different structural models of a molecule. In such a comparative analysis, the identification of the part of the molecule that is conformationally invariant with respect to a set of conformers is a critical step, as the corresponding subset of atoms constitutes the reference for subsequent analysis for example by least-squares superposition. A method is presented that categorizes atoms in a molecule as either conformationally invariant or flexible by automatic analysis of an ensemble of conformers (e.g. crystal structures from different crystal forms or molecules related by non-crystallographic symmetry). Different levels of coordinate precision, both for different models and for individual atoms, are taken explicitly into account via a modified form of Cruickshank's DPI [Cruickshank (1999), Acta Cryst. D55, 583,601] and are propagated into error-scaled difference distance matrices [Schneider (2000), Acta Cryst. D56, 715,721]. All pairwise error-scaled difference distance matrices are then analysed simultaneously using a genetic algorithm. The algorithm has been tested on several well known examples and has been found to converge rapidly to reasonable results using a standard set of parameters. In addition to the description of the algorithm, a criterion is suggested for testing the identity of two three-dimensional models within experimental error without any explicit superposition. [source] Pharmacokinetics of budesonide and formoterol administered via a series of single-drug and combination inhalers: four open-label, randomized, crossover studies in healthy adultsBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 7 2008Annika Eklund Abstract Objective: To investigate the pharmacokinetics of budesonide and formoterol administered concomitantly in healthy adults. Methods: Three single-dose, open-label crossover studies (n=28 each) were conducted (Study I: budesonide pMDI, formoterol DPI, budesonide pMDI+formoterol DPI; Study II: budesonide/formoterol pMDI, budesonide pMDI+formoterol DPI; Study III: budesonide/formoterol pMDI [three budesonide formulation strengths; constant formoterol]). Study IV (n=28) assessed steady state pharmacokinetics (budesonide/formoterol pMDI [two/four inhalations twice daily, 5-day treatment; four inhalations, single-dose]). Results: Study I: no pharmacokinetic interactions were observed between budesonide and formoterol. Study II: AUC ratios were 97.9% (budesonide) and 82.2% (formoterol) (budesonide/formoterol pMDI versus budesonide pMDI+formoterol DPI). Study III: formoterol AUC was comparable across budesonide/formoterol pMDI formulation strengths; budesonide AUC increased with formulation strength in proportion to fine particle dose. Study IV: dose proportionality was demonstrated for budesonide (AUC ratio, 104.3%) and suggested for formoterol (AUC ratio, 117.6%) with budesonide/formoterol pMDI (steady state); budesonide and formoterol AUC was higher with repeated versus single-dose budesonide/formoterol pMDI (four inhalations). Conclusions: No pharmacokinetic interactions were observed between budesonide and formoterol. Budesonide dose variation in budesonide/formoterol pMDI did not affect formoterol exposure. Steady state budesonide/formoterol pMDI dose-doubling yielded proportional increases in budesonide and formoterol exposure. Copyright © 2008 John Wiley & Sons, Ltd. [source] Relative lung and systemic bioavailability of sodium cromoglycate inhaled products using urinary drug excretion post inhalationBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 4 2002Osama Aswania Abstract The relative lung and systemic bioavailability of sodium cromoglycate following inhalation by different methods have been determined using a urinary excretion pharmacokinetic method. On three separate randomised study days, 7 days apart, subjects inhaled (i) 4×5 mg from an Intal® metered dose inhaler (MDI), (ii) 4×5 mg from an MDI attached to a large volume spacer (MDI+SP) and (iii) 20 mg from an Intal Spinhaler® (DPI). Urine samples were provided at 0, 0.5, 1, 2, 5 and 24 h post dose. The mean (S.D.) amount of sodium cromoglycate excreted in the urine during the first 30 min post inhalation was 38.1 (27.5), 222.3 (120.3) and 133.1 (92.2) ,g following MDI, MDI+SP and DPI, respectively. The mean ratio (90% confidence interval) of these amounts excreted in the urine over the first 30 min for MDI+SP vs MDI, DPI vs MDI and MDI+SP vs DPI was 801.0 (358.0, 1244; p<0.002)%, 457.0 (244.0, 670.0; p<0.02)% and 262.4 (110.2, 414.5)%, respectively. Similarly for the 24 h cumulative amount of sodium cromoglycate excreted over the 24 h post inhalation the ratios were 375.4 (232.9, 517.9; p<0.005)%, 287.5 (183.4, 391.6; p<0.02)% and 211.4 (88.3, 334.5)%, respectively. The results highlight better lung deposition of sodium cromoglycate from a metered dose inhaler attached to a large volume spacer. Copyright © 2002 John Wiley & Sons, Ltd. [source] Trade policy mix: IPR protection and R&D subsidiesCANADIAN JOURNAL OF ECONOMICS, Issue 3 2006Moonsung Kang Abstract This paper analyses strategic R&D policy under circumstances where intellectual property rights protection resulting from firms' R&D investment is not perfect. By examining policy choices wherein a government chooses both R&D subsidies and IPR protection levels simultaneously, we show that it is optimal for a government to adopt sufficiently weak IPR protection and to subsidize R&D investments of domestic firms. Inducing R&D investment of foreign rival firms will increase the profits of domestic firms. Ce mémoire analyse une politique stratégique de R&D quand la protection des droits de propriété intellectuelle (DPI) qui découlent d'investissements en R&D par les entreprises est imparfaite. En examinant les choix de politiques par lesquels un gouvernement définit simultanément les niveaux de subventions pour la R&D et de protection de la propriété intellectuelle, on montre qu'il est optimal pour un gouvernement d'adopter une protection des DPI suffisamment faible et de subventionner les investissements en R&D des entreprises nationales. Susciter un accroissement des investissements en R&D des entreprises étrangères rivales devrait augmenter les profits des entreprises nationales. [source] NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2005Qing Liu Abstract Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival. Copyright © 2004 John Wiley & Sons, Ltd. [source] Blockade of superoxide generation prevents high-affinity immunoglobulin E receptor-mediated release of allergic mediators by rat mast cell line and human basophilsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2002T. Yoshimaru Summary Background Previous studies have shown that rat peritoneal mast cells and mast cell model rat basophilic leukaemia (RBL-2H3) cells generate intracellular reactive oxygen species (ROS) in response to antigen challenge. However, the physiological significance of the burst of ROS is poorly understood. Objective The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems. Methods RBL-2H3 cells were directly stimulated with anti-rat Fc,RI ,-subunit monoclonal antibody (mAb). For the analysis of human cell system, leucocytes were isolated by dextran sedimentation from healthy volunteers or from patients, and challenged either with anti-human Fc,RI mAb or with the relevant antigens. Superoxide generation was determined by chemiluminescence-based methods. The releases of histamine and leukotrienes (LT)s were determined by enzyme-linked immunosorben assay (ELISA). Results Cross-linking of Fc,RI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-Fc,RI mAb resulted in a rapid generation of superoxide anion, as determined by chemiluminescence using superoxide-specific probes. Similarly, leucocytes from patients generated superoxide anion in response to the challenge with the relevant allergen but not with the irrelevant allergen. Furthermore, diphenyleneiodonium (DPI), a well-known inhibitor of flavoenzymes suppressed the superoxide generation and the release of histamine and LTC4 induced by the anti-Fc,RI mAb or by allergen in parallel. Conclusion These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon Fc,RI cross-linking either by antibody or by allergen challenge and that blockade of the generation prevents the release of allergic mediators. The findings strongly support the role of superoxide generation in the activation of mast cells and basophils under both physiological and pathological conditions. The findings suggest that drugs regulating the superoxide generation have potential therapeutic use for allergic disorders. [source] PIOGLITAZONE INHIBITS HOMOCYSTEINE-INDUCED MIGRATION OF VASCULAR SMOOTH MUSCLE CELLS THROUGH A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ,-INDEPENDENT MECHANISMCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2008Li Li SUMMARY 1Peroxisome proliferator-activated receptor (PPAR)-, agonists have been demonstrated to exert protective effects against homocysteine (Hcy)-induced pathogenesis. However, the effects of PPAR-, agonists on Hcy-induced migration are unknown. In the present study, we examined the effect of pioglitazone on the migration of vascular smooth muscle cells (VSMC) induced by Hcy and the possible mechanism involved. 2Vascular smooth muscle cells were isolated from the thoracic aortas of male Sprague-Dawley rats. The migration of VSMC was examined using a transwell technique. The generation of intracellular reactive oxygen species (ROS) was measured using the ROS-sensitive fluoroprobe 2,,7,-dichlorodihydrofluorescein diacetate. The activity of NAD(P)H oxidase was assessed by lucigenin enhanced chemiluminescence. Activation of p38 mitogen-activated protein kinase (MAPK) was determined by western blotting. 3The results showed that pioglitazone dose-dependently inhibited the migration of VSMC induced by Hcy. This was not reversed by the PPAR-, antagonist GW9662. In addition, pretreatment with the NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), the free radical scavenger N -acetylcysteine and the p38 MAPK inhibitor SB202190 blocked Hcy-induced VSMC migration. Furthermore, we observed that pioglitazone suppressed Hcy-induced intracellular ROS production; similar effects were observed with DPI and NAC. Pioglitazone attenuated Hcy-induced activation of NAD(P)H oxidase. Moreover, pioglitazone blocked Hcy-induced p38 MAPK phosphorylation; similar effects were observed for DPI, NAC and SB202190. 4The data demonstrate that pioglitazone inhibits Hcy-induced VSMC migration that is independent of PPAR-,. Furthermore, part of the biological effect of pioglitazone involves a decrease in the levels of NAD(P)H oxidase derived-ROS and p38 MAPK activation. [source] PHYSICAL PROPERTIES OF TWO POPULAR INDIAN POTATO VARIETIESJOURNAL OF FOOD PROCESS ENGINEERING, Issue 4 2006D.K. SINGH ABSTRACT Physical properties of potatoes, often required for analyzing heat and mass transfer during cooling and storage applications, were determined for the Kufri Jyoti and Kufri Sinduri varieties. The average values of linear dimensions, Dp, Sp, Ra, mass, ,, Acand A of these two varieties were measured. The Kufri Jyoti was found to have comparatively more variations in a, b and c than the Kufri Sinduri. The average values of Dp, Sp, Ra, ,, Acand A for the Kufri Jyoti and Kufri Sinduri were found to be 4.647 and 3.692 cm; 82.45 and 86.65%; 82.99 and 91.78%; 1113.3 and 1092.1 kg/m3; 30.86 and 12.10 cm2; and 79.05 and 47.10 cm2, respectively. The average values of ,, Dv, average Dpi, ,and bulk density for the Kufri Jyoti and Kufri Sinduri in a commercially used 50-kg gunny bag were found to be 0.42 ± 0.01 and 0.43 ± 0.008; 4.998 and 4.056 cm, 4.817 ± 0.534 and 3.922 ± 0.211 cm, 0.58 and 0.57; and 647.6 and 622.5 kg/m3, respectively. Relationships were also established for calculating the A of potato as a function of volume and weight. [source] PDGF stimulates the massive expansion of glial progenitors in the neonatal forebrainGLIA, Issue 16 2009M. C. Assanah Abstract Platelet-derived growth factor (PDGF) plays a major role in regulating migration, proliferation, and differentiation of glial progenitors during normal brain development and in the abnormal proliferation and dispersion that drives the formation of malignant gliomas. To further explore the relationship between PDGF's effects on normal glial progenitors and its role in the formation of gliomas, we infected progenitor cells in the subventricular zone (SVZ) of the lateral ventricle of neonatal rat pups with a retrovirus that expresses PDGF and green fluorescent protein (GFP). At 3 days post-injection (dpi), a proliferation of PDGFR,+ progenitors was seen in the SVZ and white matter around the injection site and by 10 dpi the animals had large diffusely infiltrating tumors that resembled glioblastomas. The tumors contained a massive proliferation of both infected and uninfected PDGFR,+ progenitors, suggesting that PDGF was driving tumor formation via both autocrine and paracrine signaling. Rats co-injected with two retroviruses (one that expresses PDGF-IRES-DSRED and one that expresses only GFP) formed tumors that contained a mixture of DSRED+ cells (PDGF producers) and GFP+ cells (recruited progenitors). Time-lapse microscopy of slice cultures confirmed that both DSRED+ and GFP+ cells were highly migratory and proliferative. Furthermore, adding exogenous PDGF to slice cultures generated from nontumor-bearing brains (injected with control GFP retrovirus only) stimulated the migration and proliferation of GFP+ progenitors. These findings reveal the inherent growth factor responsiveness and tumorigenic potential of PDGFR,+ progenitors and highlight the importance of paracrine signaling in stimulating glioma growth and infiltration. © 2009 Wiley-Liss, Inc. [source] Experimental hepatitis A virus (HAV) infection in cynomolgus monkeys (Macaca fascicularis): evidence of active extrahepatic site of HAV replicationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 1 2010Luciane A. Amado Summary This work studied the replication sites of hepatitis A virus (HAV) in cynomolgus monkeys (Macaca fascicularis) after intravenous inoculation. The cynomolgus monkeys were inoculated with the Brazilian hepatitis A virus strain (HAF-203). Monkeys were euthanized on days 15, 30, 45 and 60 postinoculation (pi). Liver samples, submandibular salivary gland, mesenteric lymph node and tonsils were removed for virological and pathological evaluation. Immunofluorescence analyses on liver and salivary gland sections using confocal laser scanning microscopy revealed the presence of HAV antigen (HAV Ag). The presence of HAV genome was monitored by real-time PCR. The HAV RNA was detected at 7 days postinoculation (dpi), concomitantly in serum, saliva and faeces. The highest HAV viral load was observed in faeces at 15 dpi (105 copies/ml), followed by serum viral load of 104 copies/ml at 20 dpi and saliva viral load of 103 copies/ml at 7 dpi. The animals showed first histological and biochemical signs of hepatitis at 15 dpi. The HAV antigen (Ag) was present from day 7 until day 60 pi in the liver and salivary glands. The HAV replicative intermediate was also detected in the liver (4.5 × 104 copies/mg), salivary glands (1.9 × 103 copies/mg), tonsils (4.2 × 101 copies/mg) and lymph nodes (3.4 × 101 copies/mg). Our data demonstrated that the salivary gland as an extrahepatic site of early HAV replication could create a potential risk of saliva transmitted infection. In addition, the cynomolgus monkey was confirmed as a suitable model to study the pathogenesis of HAV human infection. [source] Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virusJOURNAL OF FISH DISEASES, Issue 5 2006S Kerbart Boscher Abstract Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post-infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout. [source] The effect of temperature and salinity on the settlement and survival of copepodids of Lepeophtheirus salmonis (Krøyer, 1837) on Atlantic salmon, Salmo salar L.JOURNAL OF FISH DISEASES, Issue 5 2000C S Tucker The effects of temperature and salinity on the settlement, subsequent survival and development of the copepodids of Lepeophtheirus salmonis on Atlantic salmon were investigated experimentally. There was a significantly greater settlement and survival of copepodids at 10 days post-infection (dpi) at 12 °C compared with at 7 °C at a constant salinity of 34,. Development of L. salmonis was also more rapid at 12 °C. Settlement was significantly greater at a salinity of 34, than at 24,. In one experiment, survival at 10 dpi was significantly greater at 34,; however, a second experiment found that there was no significant difference between the two saline levels. This may have been because of a rise in water temperature for 2 dpi, which appears to have overridden the effect of low salinity. Development of L. salmonis was more rapid at 34,. Copepodids settled on all of the external surfaces of the salmon, although the proportion on different surfaces varied between experiments. The gills, particularly at low temperatures, the body surface, and the pectoral and dorsal fins were especially favoured. [source] Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinosMEDICAL AND VETERINARY ENTOMOLOGY, Issue 1 2002J. T. Paweska Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV-1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field-collected Culicoides were fed on sheep blood containing 7.5 log10TCID50/mL of BTV-1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK-21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ,,3 log10 TCID50) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4,10 post-infection (dpi), at 15,30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0,13.7% and 0,46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4,10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV-1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa. [source] Histopathology and PR-protein markers provide insight into adult plant resistance to stripe rust of wheatMOLECULAR PLANT PATHOLOGY, Issue 2 2008JENNIFER MOLDENHAUER SUMMARY Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a serious disease of wheat. The spring wheat cultivar Kariega expresses complete adult plant resistance to stripe rust, whereas Avocet S is susceptible. In former studies, quantitative trait loci (QTL) analysis of doubled haploid lines derived from a Kariega × Avocet S cross revealed two major QTL (QYr.sgi-7D and QYr.sgi-2B.1) and two minor QTL (QYr.sgi-1A and QYr.sgi-4A.1) responsible for the adult resistance of Kariega in the field. Avocet S contains none of these QTL. In the present study, stripe rust development was compared, by means of fluorescence and confocal laser scanning microscopy, in flag leaves of Kariega, Avocet S and six doubled haploid (DH) lines, containing all four, none or one QTL. Depending on the QTL present, the infection types of the DH lines ranged from resistant to fully susceptible. No differences in fungal growth were observed during the first 5 days post inoculation (dpi), whereas the mean length of the fungal colonies started to differ at 6 dpi. Interestingly, MP 51 carrying QYr.sgi-7D responded with lignification to the fungal growth without restricting it, whereas MP 35 containing QYr.sgi-2B.1 did not show lignified host tissue, but fungal growth was restricted. RT PCR experiments with sequences of pathogenesis-related (PR) proteins resulted in a slightly stronger induction of PR 1, 2 and 5, known markers for the hypersensitive reaction, and peroxidases in MP 51, whereas a second band for chitinases was detected in MP 35 only. [source] | |||||||||||||||||||