A comparative assessment of potential components of partial disease resistance to Fusarium head blight detected in a detached leaf assay of wheat, barley and oats.

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.

Predictors of therapy resistant asthma: outcome of a systematic evaluation protocol

Background: It has been suggested that asthmatic subjects with persisting symptoms despite adequate maintenance therapy should be systematically evaluated to identify factors contributing to poor control. The aims of this study were to examine the prevalence of these factors in a cohort of sequentially referred poorly controlled asthmatics, and to determine if any factor or combination of factors predicted true therapy resistant asthma (TRA).

Methods: Patients were evaluated using a systematic evaluation protocol including induced sputum analysis, psychiatric assessment, ear, nose and throat examination, pulmonary function testing, high resolution CT scan of the thorax, and 24 hour dual probe ambulatory oesophageal pH monitoring; any identified provoking factor was treated. Asthma was managed according to BTS guidelines.

Results: Of 73 subjects who completed the assessment, 39 responded to intervention and 34 had TRA. Subjects with TRA had a greater period of instability, a higher dose of inhaled steroids at referral, more rescue steroid use, and a lower best percentage forced expiratory volume in 1 second (FEV1%). Oesophageal reflux, upper airway disease, and psychiatric morbidity were common (57%, 95%, 49%, respectively) but were not more prevalent in either group. Using multivariate logistic regression analysis, inhaled steroid dose >2000 µg BDP, previous assessment by a respiratory specialist, and initial FEV1% of <70% at referral predicted a final diagnosis of TRA.

Conclusions: In poorly controlled asthmatics there is a high prevalence of co-morbidity, identified by detailed systematic assessment, but no difference in prevalence between those who respond to intervention and those with TRA. Targeted treatment of identified co-morbidities has minimal impact on asthma related quality of life in those with therapy resistant disease.

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(LyS(37)PAL) and N-AcGIP(LyS(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25 nmol kg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(LyS(37)PAL) or N-AcGIP(LyS37PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP. (c) 2006 Elsevier Inc. All rights reserved.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Resistance in Fasciola hepatica to triclabendazole (Fasinex) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the [beta]-tubulin molecule. Sequencing of [beta]-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 [mu]g/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Components of resistance to Fusarium head blight in wheat detected in a seed germination assay with Microdochium majus and the relationship to FHB disease development and mycotoxin accumulation from Fusarium graminearum infection.

Components of partial disease resistance (PDR) to fusarium head blight (FHB), detected in a seed-germination assay, were compared with whole-plant FHB resistance of 30 USA soft red winter wheat entries in the 2002 Uniform Southern FHB Nursery. Highly significant (P <0·001) differences between cultivars in the in vitro seed-germination assay inoculated with Microdochium majus were correlated to FHB disease incidence (r = -0·41; P <0·05), severity (r = -0·47; P <0·01), FHB index (r = -0·46; P <0·01), damaged kernels (r = -0·52; P <0·01), grain deoxynivalenol (DON) concentration (r = -0·40; P <0·05) and incidence/severity/kernel-damage index (ISK) (r = -0·45; P <0·01) caused by Fusarium graminearum. Multiple linear regression analysis explained a greater percentage of variation in FHB resistance using the seed-germination assay and the previously reported detached-leaf assay PDR components as explanatory factors. Shorter incubation periods, longer latent periods, shorter lesion lengths in the detached-leaf assay and higher germination rates in the seed-germination assay were related to greater FHB resistance across all disease variables, collectively explaining 62% of variation for incidence, 49% for severity, 56% for F. graminearum-damaged kernels (FDK), 39% for DON and 59% for ISK index. Incubation period was most strongly related to disease incidence and the early stages of infection, while resistance detected in the seed germination assay and latent period were more strongly related to FHB disease severity. Resistance detected using the seed-germination assay was notable as it related to greater decline in the level of FDK and a smaller reduction in DON than would have been expected from the reduction in FHB disease assessed by visual symptoms.