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Selected AbstractsResistance exercise increases leg muscle protein synthesis and mTOR signalling independent of sexACTA PHYSIOLOGICA, Issue 1 2010H. C. Dreyer Abstract Aim:, Sex differences are evident in human skeletal muscle as the cross-sectional area of individual muscle fibres is greater in men than in women. We have recently shown that resistance exercise stimulates mammalian target of rapamycin (mTOR) signalling and muscle protein synthesis in humans during early post-exercise recovery. Therefore, the aim of this study was to determine if sex influences the muscle protein synthesis response during recovery from resistance exercise. Methods:, Seventeen subjects, nine male and eight female, were studied in the fasted state before, during and for 2 h following a bout of high-intensity leg resistance exercise. Mixed muscle protein fractional synthetic rate was measured using stable isotope techniques and mTOR signalling was assessed by immunoblotting from repeated vastus lateralis muscle biopsy samples. Results:, Post-exercise muscle protein synthesis increased by 52% in the men and by 47% in the women (P < 0.05) and was not different between groups (P > 0.05). Akt phosphorylation increased in both groups at 1 h post-exercise (P < 0.05) and returned to baseline during 2 h post-exercise with no differences between groups (P > 0.05). Phosphorylation of mTOR and its downstream effector S6K1 increased significantly and similarly between groups during post-exercise recovery (P < 0.05). eEF2 phosphorylation decreased at 1- and 2 h post-exercise (P < 0.05) to a similar extent in both groups. Conclusion:, The contraction-induced increase in early post-exercise mTOR signalling and muscle protein synthesis is independent of sex and appears to not play a role in the sexual dimorphism of leg skeletal muscle in young men and women. [source] Influence of concurrent exercise or nutrition countermeasures on thigh and calf muscle size and function during 60 days of bed rest in womenACTA PHYSIOLOGICA, Issue 2 2007T. A. Trappe Abstract Aim:, The goal of this investigation was to test specific exercise and nutrition countermeasures to lower limb skeletal muscle volume and strength losses during 60 days of simulated weightlessness (6° head-down-tilt bed rest). Methods:, Twenty-four women underwent bed rest only (BR, n = 8), bed rest and a concurrent exercise training countermeasure (thigh and calf resistance training and aerobic treadmill training; BRE, n = 8), or bed rest and a nutrition countermeasure (a leucine-enriched high protein diet; BRN, n = 8). Results:, Thigh (quadriceps femoris) muscle volume was decreased (P < 0.05) in BR (,21 ± 1%) and BRN (,24 ± 2%), with BRN losing more (P < 0.05) than BR. BRE maintained (P > 0.05) thigh muscle volume. Calf (triceps surae) muscle volume was decreased (P < 0.05) to a similar extent (P > 0.05) in BR (,29 ± 1%) and BRN (,28 ± 1%), and this decrease was attenuated (P < 0.05) in BRE (,8 ± 2%). BR and BRN experienced large (P < 0.05) and similar (P > 0.05) decreases in isometric and dynamic (concentric force, eccentric force, power and work) muscle strength for supine squat (,19 to ,33%) and calf press (,26 to ,46%). BRE maintained (P > 0.05) or increased (P < 0.05) all measures of muscle strength. Conclusion:, The nutrition countermeasure was not effective in offsetting lower limb muscle volume or strength loss, and actually promoted thigh muscle volume loss. The concurrent aerobic and resistance exercise protocol was effective at preventing thigh muscle volume loss, and thigh and calf muscle strength loss. While the exercise protocol offset ,75% of the calf muscle volume loss, modification of this regimen is needed. [source] Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic ratsDIABETES OBESITY & METABOLISM, Issue 2 2009A. K. Dhalla Introduction and Aims:, Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT-3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary-induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT-3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. Results:, ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC50 values for forskolin-stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 ± 60 ,M) compared with SD rats (332 ± 38 ,M). EC50 values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol-stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 ± 23 ,M) compared with SD rats (343 ± 27 ,M). Insulin inhibited lipolysis in adipocytes from SD rats with an IC50 value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC50 values for CVT-3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT-3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT-3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT-3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A1 receptors relative to ,-actin expression in adipocytes from SD (0.98 ± 0.2) and ZDF rats (0.99 ± 0.3). Conclusion:, The antilipolytic effects of CVT-3619 appear to be independent of insulin resistance and animal model. [source] Inhaled insulin as adjunctive therapy in subjects with type 2 diabetes failing oral agents: a controlled proof-of-concept studyDIABETES OBESITY & METABOLISM, Issue 5 2006M. Hausmann Aim:, This controlled proof-of-concept study investigated inhaled insulin (INH) as adjunctive therapy to existing oral antidiabetic agents in subjects with type 2 diabetes. Methods:, Twenty-four subjects with type 2 diabetes [19 men and 5 women, 56.1 ± 6.6 years, body mass index 32.7 ± 4.2 kg/m2, glycosylated haemoglobin (HbA1c) 8.4 ± 0.8% (mean ± s.d.)] inadequately controlled by metformin and/or sulfonylureas were randomized to receive additional therapy with either INH administered preprandially using a metered-dose inhaler (MDI), or insulin glargine (GLA) injected subcutaneously at bedtime for 4 weeks. Both inhaled and injected insulin doses were titrated to predefined blood glucose (BG) targets. Results:, INH and GLA improved metabolic control to a similar extent. Mean daily BG decreased by 2.8 mmol/l in the INH group (p < 0.001) and by 2.4 mmol/l in the GLA group (p < 0.001). Accordingly, fasting BG (,2.7 vs. ,3.6 mmol/l for INH vs. GLA), preprandial- and 2-h postprandial BG, HbA1c (,1.23 vs. ,1.05%), body weight (,1.9 vs. ,2.3 kg) and serum fructosamine were similarly and significantly reduced in both groups (p < 0.05). Triglycerides decreased significantly with INH (,1.15 ,mol/l; p < 0.001) but not with GLA [,0.52 ,mol/l; not significant (NS)]. Incidence rates of adverse events did not differ significantly, and there were no indications of respiratory tract irritation. Conclusions:, In subjects with type 2 diabetes inadequately controlled by oral agents, preprandial administration of INH delivered by a MDI provided a comparable metabolic control to bedtime GLA and did not show any safety concerns during a 4-week treatment. These results warrant a more extensive investigation of preprandial treatment with INH in longer term studies. [source] Beneficial effects of C-peptide on incipient nephropathy and neuropathy in patients with Type 1 diabetes mellitusDIABETIC MEDICINE, Issue 3 2000B. -L. Summary Aims Recent studies have indicated that proinsulin C-peptide shows specific binding to cell membrane binding sites and may exert biological effects when administered to patients with Type 1 diabetes mellitus. This study was undertaken to determine if combined treatment with C-peptide and insulin might reduce the level of microalbuminuria in patients with Type 1 diabetes and incipient nephropathy. Methods Twenty-one normotensive patients with microalbuminuria were studied for 6 months in a double-blind, randomized, cross-over design. The patients received s.c. injections of either human C-peptide (600 nmol/24 h) or placebo plus their regular insulin regimen for 3 months. Results Glycaemic control improved slightly during the study and to a similar extent in both treatment groups. Blood pressure was unaltered throughout the study. During the C-peptide treatment period, urinary albumin excretion decreased progressively on average from 58 ,g/min (basal) to 34 ,g/min (3 months, P < 0.01) and it tended to increase, but not significantly so, during the placebo period. The difference between the two treatment periods was statistically significant (P < 0.01). In the 12 patients with signs of autonomic neuropathy prior to the study, respiratory heart rate variability increased by 21 ± 9% (P < 0.05) during treatment with C-peptide but was unaltered during placebo. Thermal thresholds were significantly improved during C-peptide treatment in comparison to placebo (n = 6, P < 0.05). Conclusion These results indicate that combined treatment with C-peptide and insulin for 3 months may improve renal function by diminishing urinary albumin excretion and ameliorate autonomic and sensory nerve dysfunction in patients with Type 1 diabetes mellitus. [source] Lactating Females Do Not Discriminate Between Their Own Young and Unrelated Pups in the Communally Breeding Rodent, Octodon degusETHOLOGY, Issue 9 2006Luis A. Ebensperger Females in numerous rodent species engage in communal nesting and breeding, in which they share one or more nests to rear their young. A potential cost of communal nesting and breeding is that mothers divert resources to unrelated offspring. One way mothers could avoid this cost is to recognize and favour their own young over unrelated offspring when allocating maternal effort. We assessed whether female degus (Octodon degus), a communally nesting and breeding caviomorph rodent, discriminate between their own and unrelated offspring during lactation. Female degus previously have been shown to distinguish between their own and unrelated pups when exposed to odours from both. We measured pup discrimination based on differences in the retrieval behaviour of females that were in early or intermediate lactation directed towards their own and unrelated offspring; offspring presented were of similar or different age. Before any event of pup retrieval, lactating females spent similar amounts of time and interacted to a similar extent with their own and unrelated pups. During pup retrieval, all lactating females transported both pups to the nest. Neither relatedness to pups, nor pup-age differences, influenced the order in which pups were retrieved to the nest. Dams waited similar amounts of time before retrieving the first pup when the first transported young was their own or unrelated. Likewise, females waited similar amounts of time before retrieving the second pup when the pup transported first was their own or unrelated. The time between first and second pup transport events was longer when dams were in early when compared with intermediate lactation, but only when pups were of similar age. All experimental subjects nursed unrelated pups after they were retrieved. Collectively, our results do not support the hypothesis that communally breeding female degus use their recognition ability to discriminate against unrelated offspring in favour of their own young. [source] Lifestyle intervention in individuals with normal versus impaired glucose toleranceEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2007S. Schäfer Abstract Background, Lifestyle intervention is effective in the prevention of type 2 diabetes in individuals with impaired glucose tolerance (IGT). It is currently unknown whether it has beneficial effects on metabolism to a similar extent, in individuals with normal glucose tolerance (NGT) compared to individuals with IGT. Materials and methods, Data from 181 subjects (133 with NGT and at risk for type 2 diabetes and 48 with IGT) who participated in the Tuebingen Lifestyle Intervention Program with increase in physical activity and decrease in caloric intake were included into this study. Body fat distribution was quantified by whole-body magnetic resonance (MR) tomography and liver fat and intramyocellular fat by 1H-MR spectroscopy. Insulin sensitivity was estimated from an oral glucose tolerance test (OGTT). Results, After 9 ± 2 months of follow-up, the diagnosis of IGT was reversed in 24 out of 48 individuals. Only 14 out of 133 participants with NGT developed IGT. Body weight decreased in both groups by 3% (both P < 0·0001). Two-hour glucose concentrations during an OGTT decreased in individuals with IGT (,14%, P < 0·0001) but not with NGT (+2%, P = 0·66). Insulin sensitivity increased both in individuals with IGT (+9%, P = 0·04) and NGT (+17%, P < 0·0001). Visceral fat (,8%, P = 0·006), liver fat (,28%, P < 0·0001) and intramyocellular fat (,15%, P = 0·006) decreased in participants with IGT. In participants with NGT these changes were significant for visceral fat (,16%, P < 0·0001) and liver fat (,35%, P < 0·0001). Conclusions, Moderate weight loss under a lifestyle intervention with reduction in total, visceral and ectopic fat and increase in insulin sensitivity improves glucose tolerance in individuals with IGT but not with NGT. In individuals with NGT, the beneficial effects of a lifestyle intervention on fat distribution and insulin sensitivity possibly prevent future deterioration in glucose tolerance. [source] Broad T cell immunity to the LcrV virulence protein is induced by targeted delivery to DEC-205/CD205-positive mouse dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Yoonkyung Do Abstract There is a need for a more efficient vaccine against the bacterium Yersinia pestis, the agent of pneumonic plague. The F1-LcrV (F1-V) subunit vaccine in alhydrogel is known to induce humoral immunity. In this study, we utilized DC to investigate cellular immunity. We genetically engineered the LcrV virulence protein into the anti-DEC-205/CD205 mAb and thereby targeted the conjugated protein directly to mouse DEC-205+ DC in situ. We observed antigen-specific CD4+ T cell immunity measured by intracellular staining for IFN-, in three different mouse strains (C57BL/6, BALB/c, and C3H/HeJ), while we could not observe such T cell responses with F1-V vaccine in alhydrogel. Using a peptide library for LcrV protein, we identified two or more distinct CD4+ T cell mimetopes in each MHC haplotype, consistent with the induction of broad immunity. When compared to nontargeted standard protein vaccine, DC targeting greatly increased the efficiency for inducing IFN-,-producing T cells. The targeted LcrV protein induced antibody responses to a similar extent as the F1-V subunit vaccine, but Th1-dependent IgG2a and IgG2c isotypes were observed only after anti-DEC-205:LcrV mAb immunization. This study sets the stage for the analysis of functional roles of IFN-,-producing T cells in Y.,pestis infection. [source] Evidence for RPE65-independent vision in the cone-dominated zebrafish retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Helia B. Schonthaler Abstract An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11- cis -retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all- trans to 11- cis -retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all- trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11- cis -retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11- cis -retinal for cone vision. [source] SHORT COMMUNICATION Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKCEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2002Yaron Seroussi Abstract We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl- sn -glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells. [source] Inhibition of scratching behaviour caused by contact dermatitis in histidine decarboxylase gene knockout miceEXPERIMENTAL DERMATOLOGY, Issue 3 2005M. Seike Abstract:, A neuronal system dedicated to itch consists of primary afferent and spinothalamic projection neurons. Histamine is thought to be one of the main mediators for the transmission of itch sensation. However, there are little available information on the role of histamine in scratching behaviour and sensory transmission of atopic dermatitis and chronic eczema. In the present study, the role of histamine in scratching behaviour and neural conduction of sensation in the chronic eczema model was investigated by using l-histidine decarboxylase (HDC) gene knockout mice lacking histamine. The chronic contact dermatitis was induced with daily application of diphenylcyclopropenone (DCP) on a hind paw of HDC (+/+) and HDC (,/,) mice for 2 months. The observation of scratching behaviour and the hot-plate test were performed in both mice. Histological studies were performed in the skin and spinal cord tissues. Histological examination revealed that both HDC (+/+) and HDC (,/,) mice displayed the similar extent of inflammatory cell infiltration, hyperplastic epidermis and newly spreading of neuronal processes in the skin tissue. Scratching behaviour was exclusively induced in HDC (+/+) mice, whereas it was barely observed in HDC (,/,) mice. The expression of c-Fos was specifically upregulated in HDC (+/+) mice in lamina I of the spinal dorsal horn following repeated DCP application. Scratching behaviour in chronic contact dermatitis in mice was thought mainly mediated with histamine. The afferent pathway of sensation in chronic contact dermatitis model may connect with the central nervous system through lamina I of the spinal dorsal horn. [source] Angiotensin-(1,7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating STAT3 and STAT5a/b phosphorylation and inhibiting angiotensin II-stimulated ERK1/2 and Rho kinase activityEXPERIMENTAL PHYSIOLOGY, Issue 5 2008Jorge F. Giani Angiotensin (ANG) II contributes to cardiac remodelling by inducing the activation of several signalling molecules, including ERK1/2, Rho kinase and members of the STAT family of proteins. Angiotensin-(1,7) is produced in the heart and inhibits the proliferative actions of ANG II, although the mechanisms of this inhibition are poorly understood. Accordingly, in the present study we examined whether ANG-(1,7) affects the ANG II-mediated activation of ERK1/2 and Rho kinase, STAT3 and STAT5a/b in rat heart in vivo. We hypothesized that ANG-(1,7) inhibits these growth-promoting pathways, counterbalancing the trophic action of ANG II. Solutions of normal saline (0.9% NaCl) containing ANG II (8 pmol kg,1) plus ANG-(1,7) in increasing doses (from 0.08 to 800 pmol kg,1) were administered via the inferior vena cava to anaesthetized male Sprague,Dawley rats. After 5 min, hearts were removed and ERK1/2, Rho kinase, STAT3 and STAT5a/b phosphorylation was determined by Western blotting using phosphospecific antibodies. Angiotensin II stimulated ERK1/2 and Rho kinase phosphorylation (2.3 ± 0.2- and 2.1 ± 0.2-fold increase over basal values, respectively), while ANG-(1,7) was without effect. The ANG II-mediated phosphorylation of ERK1/2 and Rho kinase was prevented in a dose-dependent manner by ANG-(1,7) and disappeared in the presence of the Mas receptor antagonist d -Ala7 -ANG-(1,7). Both ANG II and ANG-(1,7) increased STAT3 and STAT5a/b phosphorylation to a similar extent (130,140% increase). The ANG-(1,7)-stimulated STAT phosphorylation was blocked by the AT1 receptor antagonist losartan and not by d -Ala7 -ANG-(1,7). Our results show a dual action of ANG-(1,7), that is, a stimulatory effect on STAT3 and 5a/b phosphorylation through AT1 receptors and a blocking action on ANG II-stimulated ERK1/2 and Rho kinase phosphorylation through Mas receptor activation. The latter effect could be representative of a mechanism for a protective role of ANG-(1,7) in the heart by counteracting the effects of locally generated ANG II. [source] Effect of fatty acid-binding proteins on intermembrane fatty acid transportFEBS JOURNAL, Issue 19 2000Studies on different types, mutant proteins Liposomes of different charge fixed to nitrocellulose filters were used to study the transfer of fatty acids to rat heart or liver mitochondria in the presence of fatty acid-binding protein (FABP) or albumin. [14C]Palmitate oxidation was used as a parameter. Different FABP types and heart FABP mutants were tested. The charge of the liposomes did not influence the solubilization and mitochondrial oxidation of palmitate without FABP and the amount of solubilized palmitate in the presence of FABP. Mitochondria did not show a preference for oxidation of FABP-bound palmitate over their tissue-specific FABP type. All FABP types increased palmitate oxidation by heart and liver mitochondria with neutral, positive and negative liposomes by 2.5-fold, 3.2-fold and twofold, respectively. Ileal lipid-binding protein and H-FABP mutants that do not bind fatty acid had no effect. Other H-FABP mutants had different effects, dependent on the site of mutation. The effect of albumin was similar to, but not dependent on, liposome charge. The ionic strength had only a slight effect. In conclusion, the transfer of palmitate from liposomal membranes to mitochondria was increased by all FABP types to a similar extent. The membrane charge had a large effect in contrast to the origin of the mitochondria. [source] Pluronics' influence on pseudomonad biofilm and phenazine productionFEMS MICROBIOLOGY LETTERS, Issue 1 2009Lindsay Housley Abstract Colonization of roots by Pseudomonas chlororaphis O6 (PcO6) involves root surface coverage through surface motility and biofilm formation. Root colonization and the production of antifungal phenazines are important in the ability of the bacterium to protect plants against pathogens. In this in vitro study we report that both biofilm formation and phenazine production are differentially influenced by nutrition and the presence of polyethylene oxide/polypropylene oxide triblock copolymer surfactants (Pluronics). Such surfactants are used for many purposes including agricultural formulations. Four Pluronics differing in molecular weight and in hydrophobic/hydrophilic proportions had distinct effects on biofilm formation and secondary metabolite production, although each increased surface motility, termed swarming, to a similar extent. These findings show that Pluronics had specific metabolic impacts on the bacterium, where both up- and downregulation was achieved depending on the medium and the Pluronic composition. In environmental and agricultural settings, Pluronics may have unanticipated effects on soil microorganisms, while in bioprocessing these effects may be leveraged to regulate metabolite yield. [source] Effect kinetics of desmopressin-induced platelet retention in healthy volunteers treated with aspirin or placeboHAEMOPHILIA, Issue 1 2000Lethagen Desmopressin is often used for haemostatic treatment in platelet dysfunction, but the effect kinetics of platelet responses and the mechanism of action are poorly known. This study aimed to determine the kinetics of platelet function responses induced by desmopressin in healthy volunteers treated with aspirin or placebo. Another aim was to correlate platelet responses to changes of von Willebrand factor (vWF) in plasma. We measured platelet function with a glass bead retention test, Ivy bleeding time, vWF:Ag and multimeric structure in plasma. Median baseline platelet retention was 12% (normal reference range 16,27%) during aspirin treatment and 18% during placebo. Median peak platelet retention after desmopressin was 33% during aspirin treatment and 34% during placebo. After about 3 h platelet function had returned to baseline. A second desmopressin dose after 3 h stimulated platelet retention to a similar extent as the first dose. There was no correlation between platelet responses and quantitative or qualitative changes of vWF in plasma. Platelet count did not change significantly. Thus, desmopressin's effect on platelet function lasts for about 3 h, but may be prolonged by a second dose immediately thereafter. These findings may have important clinical implications for patients with aspirin-induced platelet dysfunction undergoing surgery. [source] The angiogenic makeup of human hepatocellular carcinoma does not favor vascular endothelial growth factor/angiopoietin-driven sprouting neovascularization,,HEPATOLOGY, Issue 5 2008Wenjiao Zeng Quantitative data on the expression of multiple factors that control angiogenesis in hepatocellular carcinoma (HCC) are limited. A better understanding of the mechanisms underlying angiogenesis in HCC will improve the rational choice of anti-angiogenic treatment. We quantified gene and protein expression of members of the vascular endothelial growth factor (VEGF) and angiopoietin systems and studied localization of VEGF, its receptors VEGFR-1 and VEGFR-2, Angiopoietin (Ang)-1 and Ang-2, and their receptor, in HCC in noncirrhotic and cirrhotic livers. We employed real-time reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunohistology, and compared the outcome with highly angiogenic human renal cell carcinoma (RCC). HCC in noncirrhotic and cirrhotic livers expressed VEGF and its receptors to a similar extent as normal liver, although in cirrhotic background, VEGFR-2 levels in both tumor and adjacent tissue were decreased. Ang-1 expression was slightly increased compared with normal liver, whereas Tie-2 was strongly down-regulated in the tumor vasculature. Ang-2 messenger RNA (mRNA) levels were also low in HCCs of both noncirrhotic and cirrhotic livers, implying that VEGF-driven angiogenic sprouting accompanied by angiopoietin-driven vascular destabilization is not pronounced. In RCC, VEGF-A levels were one order of magnitude higher. At the same time, endothelially expressed Ang-2 was over 30-fold increased compared with expression in normal kidney, whereas Ang-1 expression was decreased. Conclusion: In hepatocellular carcinoma, tumor vascularization is not per se VEGF/angiopoietin driven. However, increased CD31 expression and morphological changes representative of sinusoidal capillarization in tumor vasculature indicate that vascular remodeling is taking place. This portends that therapeutic intervention of HCC at the level of the vasculature is optional, and that further studies into the molecular control thereof are warranted. (HEPATOLOGY 2008.) [source] Impairment of health-related quality of life in patients with inflammatory bowel disease: A Spanish multicenter studyINFLAMMATORY BOWEL DISEASES, Issue 5 2005Dr. F Casellas PhD Abstract Background: Inflammatory bowel disease impairs patients' perception of health and has a negative impact on health-related quality of life (HRQOL). Most studies include patients from a single hospital. This may bias limit results through the use of small patient samples and/or samples within a restricted disease spectrum. Methods: HRQOL was measured in patients with ulcerative colitis (UC) and Crohn's disease (CD) from 9 hospitals located in different geographical areas in Spain using 2 questionnaires: the Spanish version of the Inflammatory Bowel Disease Questionnaire (IBDQ) and the EuroQol. Results are expressed as medians. Results: The study included 1156 patients (528 patients with UC and 628 with CD; median age, 35 yr; slight predominance of women, 617 versus 539). HRQOL worsened in parallel with disease severity to a similar extent in both UC (IBDQ scores of 6.1, 4.7, and 4.0 for the 3 disease severity groups, respectively) and CD (IBDQ scores of 6.1, 5.0, and 4.1, respectively). A similar inverse relation between clinical activity and quality of life was observed when EuroQol preference values were used. All 5 dimensions of the IBDQ showed significantly lower scores in patients with active UC and CD than in patients in remission. The pattern of scores by IBDQ dimensions differed between patients in relapse (who scored worse on the digestive symptoms dimension) and patients in remission. Variables related with disease activity, time of evolution since diagnosis and female sex, were significantly associated with having a worse perception of HRQOL. The type of disease or geographical area of residence did not influence results on the IBDQ. Conclusions: UC and CD impair patients' HRQOL, and the degree of impairment depends on disease activity but is independent of the type of disease and place of residence. [source] Bioaccessibility studies of ferro-chromium alloy particles for a simulated inhalation scenario: A comparative study with the pure metals and stainless steelINTEGRATED ENVIRONMENTAL ASSESSMENT AND MANAGEMENT, Issue 3 2010Klara Midander Abstract The European product safety legislation, REACH, requires that companies that manufacture, import, or use chemicals demonstrate safe use and high level of protection of their products placed on the market from a human health and environmental perspective. This process involves detailed assessment of potential hazards for various toxicity endpoints induced by the use of chemicals with a minimum use of animal testing. Such an assessment requires thorough understanding of relevant exposure scenarios including material characteristics and intrinsic properties and how, for instance, physical and chemical properties change from the manufacturing phase, throughout use, to final disposal. Temporary or permanent adverse health effects induced by particles depend either on their shape or physical characteristics, and/or on chemical interactions with the particle surface upon human exposure. Potential adverse effects caused by the exposure of metal particles through the gastrointestinal system, the pulmonary system, or the skin, and their subsequent potential for particle dissolution and metal release in contact with biological media, show significant gaps of knowledge. In vitro bioaccessibility testing at conditions of relevance for different exposure scenarios, combined with the generation of a detailed understanding of intrinsic material properties and surface characteristics, are in this context a useful approach to address aspects of relevance for accurate risk and hazard assessment of chemicals, including metals and alloys and to avoid the use of in vivo testing. Alloys are essential engineering materials in all kinds of applications in society, but their potential adverse effects on human health and the environment are very seldom assessed. Alloys are treated in REACH as mixtures of their constituent elements, an approach highly inappropriate because intrinsic properties of alloys generally are totally different compared with their pure metal components. A large research effort was therefore conducted to generate quantitative bioaccessibility data for particles of ferro-chromium alloys compared with particles of the pure metals and stainless steel exposed at in vitro conditions in synthetic biological media of relevance for particle inhalation and ingestion. All results are presented combining bioaccessibility data with aspects of particle characteristics, surface composition, and barrier properties of surface oxides. Iron and chromium were the main elements released from ferro-chromium alloys upon exposure in synthetic biological media. Both elements revealed time-dependent release processes. One week exposures resulted in very small released particle fractions being less than 0.3% of the particle mass at acidic conditions and less than 0.001% in near pH-neutral media. The extent of Fe released from ferro-chromium alloy particles was significantly lower compared with particles of pure Fe, whereas Cr was released to a very low and similar extent as from particles of pure Cr and stainless steel. Low release rates are a result of a surface oxide with passive properties predominantly composed of chromium(III)-rich oxides and silica and, to a lesser extent, of iron(II,III)oxides. Neither the relative bulk alloy composition nor the surface composition can be used to predict or assess the extent of metals released in different synthetic biological media. Ferro-chromium alloys cannot be assessed from the behavior of their pure metal constituents. Integr Environ Assess Manag 2010;6:441,455. © 2009 SETAC [source] Mechanism of blood coagulation in common carp (Cyprinus carpio)INTEGRATIVE ZOOLOGY (ELECTRONIC), Issue 3 2006Shuangan LI Abstract In vitro, carp blood was anticoagulated by using MgSO4 at a final concentration of 22.2 mmol L,1 and sodium citrate at a final concentration of 11.8 mmol L,1. The coagulation times for carp plasma diluted by ion-free water (1:1), and that of carp plasma to which thrombocytes and small lymphocytes were added, were measured at 23 °C using standard methods, and then contrasted with the coagulation times for plasma obtained from chickens and rabbits. The shapes of the thrombocytes and small lymphocytes, which were either wet mounted or stained with hematoxylin and eosin, were observed under a light microscope. We found that: (i) the coagulation reaction of carp blood was significantly (P < 0.01) accelerated by the addition of ion-free water; (ii) the three types of blood cells (thrombocytes, small lymphocytes and red blood cells) promoted plasma coagulation to a similar extent (P > 0.05); (iii) in carp Mg2+ plasma and K2C2O4 plasma, the thrombocytes were usually morphologically normal, but many small lymphocytes were destroyed and became aggregated; (iv) in the citrate plasma, thrombocytes were often aggregated, but the small lymphocytes were usually morphologically normal; and (v) the coagulation time for chicken and rabbit plasma was significantly extended by adding ion-free water. [source] Remifentanil post-conditioning attenuates cardiac ischemia,reperfusion injury via , or , opioid receptor activationACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 4 2010G. T. C. WONG Background: Ischemic pre- or post-conditioning of the heart has been shown to involve opioid receptors. Remifentanil, an ultra-short-acting selective , opioid receptor agonist in clinical use, pre-conditions the rat heart against ischemia,reperfusion injury. This study investigates whether remifentanil post-conditioning is also cardioprotective. Methods: Remifentanil post-conditioning (5-min infusion at 1, 5, 10 or 20 ,g/kg/min) or ischemic post-conditioning (three cycles of a 10 s reperfusion interspersed with a 10 s ischemia) was induced in an open-chest rat heart model of ischemia and reperfusion injury, in the presence or absence of nor-binaltorphimine, naltrindole or CTOP, specific ,, , and , opioid receptor antagonists, respectively. The same sequence of experiments was repeated in the isolated heart model using the maximal protective dose of remifentanil from the dose,response studies. Results: Both ischemic and remifentanil post-conditioning reduced the myocardial infarct size relative to the control group in both models. This cardioprotective effect for both post-conditioning regimes was prevented by the prior administration of nor-binaltorphimine and naltrindole but not CTOP. The sole administration of the antagonists had no effect on the size of myocardial infarction. Conclusions: These results indicate that remifentanil post-conditioning protects the heart from ischemia,reperfusion injury to a similar extent as of ischemic post-conditioning. This protection involves , and , but not , opioid receptor activation. This drug has great potential as a clinical post-conditioning modality as it can be given in large doses without prolonged opioid-related side effects. [source] A Common SCN5A Variant Alters the Responsiveness of Human Sodium Channels to Class I Antiarrhythmic AgentsJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 4 2007MOSSAAB SHURAIH M.D. Background: The potential pathophysiological role of common SCN5A polymorphisms in cardiac arrhythmias has been increasingly recognized. However, little is known about the impact of those polymorphisms on the pharmocological response of hNav1.5 to various antiarrhythmic agents. Methods and Results: The known SCN5A polymorphism, S524Y, was studied in comparison with the wild type (WT) define the SCN5A-Q1077del variant. The ion channel gating kinetics and pharmacology were evaluated using whole-cell patch-clamp methods in HEK-293 cells. Consistent with a previous report, the basal ion channel gating kinetics of S524Y were indistinguishable from the WT. Quinidine (20 ,M) caused similar extent of tonic block reduction of sodium currents at ,120 mV in WT and S524Y. Surprisingly, quinidine (20 ,M) exerted a more use-dependent block by a 10 Hz pulse train in S524Y than in WT at 22°C (Ki: WT, 51.3 ,M; S524Y, 20.3 ,M). S524Y significantly delayed recovery from the use-dependent block, compared with the WT (,= 88.6 ± 7.9 s vs 41.9 ± 6.6 s, P < 0.005). Under more physiological conditions using a 2 Hz pulse train at 37°C, S524Y similarly enhanced the use-dependent block by quinidine. In addition, S524Y enhanced the use-dependent block by flecainide (12.5 ,M), but not by mexiletine (100 ,M). Conclusion: A common SCN5A polymorphism, S524Y, can enhance a use-dependent block by class Ia and Ic antiarrhythmic agents. Our findings may have clinical implications in pharmacological management of cardiac arrhythmias since this common SCN5A polymorphism might be a contributing factor to the variable antiarrhythmic response. [source] Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibitionAGING CELL, Issue 6 2009María Paz Gavilán Summary Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1,, ATF6, and PERK, whereas aged rats failed to induce the IRE1, and ATF6, pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of pro-apoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders. [source] The association between metabolic syndrome, microalbuminuria and impaired renal function in the general population: impact on cardiovascular disease and mortalityJOURNAL OF INTERNAL MEDICINE, Issue 4 2007K. P. Klausen Abstract. Objective:, Microalbuminuria and metabolic syndrome are both associated with cardiovascular disease (CVD). The aim of this study was to determine the potential association between numbers of components in the metabolic syndrome, different levels of microalbuminuria and renal function. We also aimed to determine the risk of death and CVD at different levels of microalbuminuria and renal function and numbers of components in the metabolic syndrome. Design:, Population-based observational follow-up study Setting:, Epidemiological research unit (Copenhagen City Heart Study). Subjects:, A total of 2696 men and women, 30,70 years of age. Baseline measures:, Urinary albumin excretion (UAE), creatinine clearance and metabolic risk factors were measured in 1992,1994. Main outcome measurements:, The participants were followed prospectively by registers until 1999,2000 with respect to CVD, and until 2004 with respect to death. Results:, We found a strong association between microalbuminuria and the metabolic syndrome: 2% with none and 18% with five metabolic risk factors had microalbuminuria (P < 0.001). No association between impaired renal function defined as creatinine clearance <60 mL min,1 and the metabolic syndrome was found. Microalbuminuria was associated with increased risk of death and CVD to a similar extend as the metabolic syndrome, irrespective of concomitant presence of metabolic syndrome (RR,2; P < 0.001). Impaired renal function was not associated with increased risk of death and CVD in subjects with the metabolic syndrome. Conclusions:, Microalbuminuria (UAE >5 ,g min,1) confers increased risk of death and CVD to a similar extent as the metabolic syndrome. [source] Constitutive high-affinity choline transporter endocytosis is determined by a carboxyl-terminal tail dileucine motifJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Fabiola M. Ribeiro Abstract Maintenance of acetylcholine synthesis depends on the effective functioning of a high-affinity sodium-dependent choline transporter (CHT1). Recent studies have shown that this transporter is predominantly localized inside the cell, unlike other neurotransmitter transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylcholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis. [source] Homeostasis of neuroactive amino acids in cultured cerebellar and neocortical neurons is influenced by environmental cuesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Helle Waagepetersen Abstract Neuronal function is highly influenced by the extracellular environment. To study the effect of the milieu on neurons from cerebellum and neocortex, cells from these brain areas were cultured under different conditions. Two sets of cultures, one neocortical and one cerebellar neurons, were maintained in media containing [U- 13C]glucose for 8 days at initial concentrations of 12 and 28 mM glucose, respectively. Other sets of cultures (8 days in vitro) maintained in a medium containing initially 12 mM glucose were incubated subsequently for 4 hr either by addition of [U- 13C]glucose to the culture medium (final concentration 3 mM) or by changing to fresh medium containing [U- 13C]glucose (3 mM) but without glutamine and fetal calf serum. 13C Nuclear magnetic resonance (NMR) spectra revealed extensive ,-aminobutyric acid (GABA) synthesis in both cultured neocortical and cerebellar neurons after maintenance in medium containing [U- 13C]glucose for 8 days, whereas no aspartate labeling was observed in these spectra. Mass spectrometry analysis, however, revealed high labeling intensity of aspartate, which was equal in the two types of neurons. Addition of [U- 13C]glucose (4 hr) on Day 8 in culture led to a similar extent of labeling of GABA in neocortical and in cerebellar cultures, but the cellular content of GABA was considerably higher in the neocortical neurons. The cellular content of alanine was similar regardless of culture type. Comparing the amount of labeling, however, cerebellar neurons exhibited a higher capacity for alanine synthesis. This is compatible with the fact that cerebellar neurons could ameliorate a low alanine content after culturing in low glucose (12 mM) by a 4-hr incubation in medium containing 3 mM glucose. A low glucose concentration during the culture period and a subsequent medium change were associated with decreases in glutathione and taurine contents. Moreover, glutamate and GABA contents were reduced in cerebellar cultures under either of these conditions. In neocortical neurons, the GABA content was decreased by simultaneous exposure to low glucose and change of medium. These conditions also led to an increase in the aspartate content in both types of cultures, although most pronounced in the neocortical neurons. Further experiments are needed to elucidate these phenomena that underline the impact of extracellular environment on amino acid homeostasis. © 2004 Wiley-Liss, Inc. [source] The importance of brain PGE2 inhibition versus paw PGE2 inhibition as a mechanism for the separation of analgesic and antipyretic effects of lornoxicam in rats with paw inflammationJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2009Dr Nobuko Futaki Abstract Objectives Lornoxicam is a non-selective cyclooxygenase inhibitor that exhibits strong analgesic and anti-inflammatory effects but a weak antipyretic effect in rat models. Our aim was to investigate the mechanism of separation of potencies or analgesic and antipyretic effecls of lornoxicam in relatioin to its effect on prostaglandin E2 (PGE2) production in the inflammatory paw and the brain. Methods A model of acute or chronic paw inflammation was induced by Freund's complete adjuvant injection into the rat paw. Lornoxicam (0.01,1 mg/kg), celecoxib (0.3,30 mg/kg) or loxoprofen (0.3,30 mg/kg) was administered orally to the rats and the analgesic and antipyretic effects were compared. The paw hyperalgesia was assessed using the Randall,Selitto test or the flexion test. Dorsal subcutaneous body temperature was measured as indicator of pyresis. After the measurement of activities, the rats were sacrificed and the PGE2 content in the paw exudate, cerebrospinal fluid or brain hypothalamus was measured by enzme-immunoassay. Key findings In a chronic model of arthritis, lornoxicam, celecoxib and loxoprofen reduced hyperalgesia with an effective dose that provides 50% inhibition (ED50) of 0.083, 3.9 and 4.3 mg/kg respectively, whereas the effective dose of these drugs in pyresis was 0.58, 0.31 and 0.71 mg/kg respectively. These drugs significantly reduced the PGE2 level in paw exudate and the cerebrospinal fluid. In acute oedematous rats, lornoxicam 0.16 mg/kg, celecoxib 4 mg/kg and loxoprofen 2.4 mg/kg significantly reduced hyperalgesia to a similar extent. On the other hand, lornnoxicam did not affect the elevated body temperature, whereas celecoxib and loxoprofen siginificantly reduced the pyrexia to almost the normal level. These drugs significantly reduced the PGE2 level in inflamed paw exudate lo almost the normal level. On the other hand, lornoxicam did not change PGE2 level in the brain hypothalamus, whereas celecoxib and loxoprofen strongly decreased it. Conclusions Lornoxicam exhibits strong analgesic but weak antipyretic effects in rats with paw inflammation. Such a separation of effects is related to its efficacy in the reduction of PGE2 levels in the paw and brain hypothalamus. [source] Acute Effects of Ethanol on Kainate Receptors in Cultured Hippocampal NeuronsALCOHOLISM, Issue 2 2000Edmar T. Costa Background: Kainate receptors are a subclass of ionotropic glutamate receptors that regulate excitability and mediate synaptic transmission and plasticity in the hippocampus. The acute effects of ethanol on these receptors are not completely understood. Methods: The acute effects of ethanol on pharmacologically isolated kainate receptor-mediated currents were studied in cultured hippocampal neurons obtained from neonatal rats. Whole-cell patch-clamp electrophysiological techniques were used for these studies. LY303070 (GYKI-53784), a potent AMPA (,-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor-selective noncompetitive antagonist, was used to isolate kainate currents. Results: Kainate receptor-mediated currents corresponded to 7% of the total non- N -methyl- d -aspartate (non-NMDA) currents in these neurons and were reduced to 24% of control values in the presence of 15 ,M lanthanum. These kainate receptor-mediated currents were significantly inhibited by ethanol concentrations of 50 mM or more. Under our recording conditions, ethanol inhibited non-NMDA receptor- and NMDA receptor-mediated currents to a similar extent as kainate receptor-mediated currents. Western blot analysis indicated that glutamate receptor-5 and -6/7 subunits, and kainic acid-2 subunits are expressed in these cultured hippocampal neurons. Conclusions: The present results suggest that kainate receptors are important targets for the actions of ethanol in the central nervous system. [source] Aspirin and salicylate inhibit colon cancer medium- and VEGF-induced endothelial tube formation: correlation with suppression of cyclooxygenase-2 expressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003M. I. Shtivelband Summary., To determine whether aspirin and salicylate suppress colon cancer cell-mediated angiogenesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation on Matrigel. Aspirin and sodium salicylate concentration-dependently inhibited human endothelial cell (EC) tube formation induced by conditioned medium collected from DLD-1, HT-29 or HCT-116 colon cancer cells. Aspirin and sodium salicylate at pharmacological concentrations were equally effective in blocking tube formation. Neutralizing antivascular endothelial growth factor (VEGF) antibodies blocked colon cancer medium-induced tube formation. VEGF receptor 2 but not receptor 1 antibodies inhibited tube formation to a similar extent as anti-VEGF antibodies. These results indicate that VEGF interaction with VEGF receptor 2 is the primary mechanism underlying colon cancer-induced angiogenesis. Aspirin or sodium salicylate inhibited VEGF-induced tube formation in a concentration-dependent manner comparable to that of inhibition of colon cancer medium-induced endothelial tube formation. It has been shown that cyclooxygenase-2 (COX-2) is pivotal in cancer angiogenesis. We found that colon cancer medium-induced COX-2 protein expression in EC and aspirin or sodium salicylate suppressed the cancer-induced COX-2 protein levels at concentrations correlated with those that suppressed endothelial tube formation. Furthermore, aspirin and sodium salicylate inhibited COX-2 expression stimulated by VEGF. These findings indicate that aspirin and other salicylate drugs at pharmacological concentrations inhibit colon cancer-induced angiogenesis which is correlated with COX-2 suppression. [source] CD40 and OX40 ligand are differentially regulated on asthmatic airway smooth muscleALLERGY, Issue 7 2009D. I. Krimmer Background:, CD40 and OX40 Ligand (OX40L) are cell-surface molecules expressed on airway smooth muscle (ASM) that can enhance inflammatory cell activation and survival. The aim of this study was to examine the effect of tumour necrosis factor-alpha (TNF-,) and interferon-gamma (IFN-,) on ASM CD40 and OX40L expression. Methods:, CD40 and OX40L expression on human ASM cells from asthmatic and nonasthmatic donors following stimulation with TNF-, and/or IFN-, was measured using cell-surface enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Involvement of signalling pathway was investigated with pharmacological inhibitors. Soluble TNF receptor levels were quantified by ELISA. Results:, Interferon-, and TNF-, synergistically increased CD40 expression to a greater extent on asthmatic than on nonasthmatic ASM. In contrast, IFN-, reduced TNF-,-induced OX40L expression to a similar extent in both cell types. TNF-, and IFN-, induced CD40 via nuclear factor-,B (NF-,B) and signal transducer and activator of transcription-3 in both cell types and modulated OX40L via NF-,B and c-Jun N terminal kinase in nonasthmatic cells. Similar effects on the induction of OX40L in asthmatic cells were seen with NF-,B, but these were not statistically significant. The reduced OX40L expression with TNF-, and IFN-, involved extracellular regulated kinase 1/2 activation. Conclusion:, Asthmatic ASM may modulate airway inflammation locally by increasing CD40 and OX40L expression in response to cytokines. IFN-, may regulate ASM pro-inflammatory actions by differentially modulating ASM CD40 and OX40L expression. [source] The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] | ||||||||||||||||||||||||||||||||||