Cysteinyl leukotriene receptor 1 is a potent regulator of the endosomal-lysosomal system in the ARPE-19 retinal pigment epithelial cell line.

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Pyramiding of transgenic immune receptors from primary and tertiary wheat gene pools improves powdery mildew resistance in the field.

Introgression of resistance genes from wild or related species is a common strategy to improve disease resistance of wheat cultivars. Pm17 is a gene that confers powdery mildew resistance in wheat. It encodes an NLR type of immune receptor and was introgressed from rye to wheat as part of the 1RS chromosome arm translocation several decades ago. So far it has not been possible to separate Pm17 from its co-introgressed rye genes due to suppressed recombination. Here we tested in the field transgenic Bobwhite wheat overexpressing Pm17 without any other rye genes. Four transgenic events showed high levels of PM17 protein accumulation, strong powdery mildew resistance, and no pleiotropic effects during three field seasons. We used a combined approach of transgene insertion and cross-breeding to generate lines co-expressing Pm17 and Pm3, or Pm17 and Pm8. Blumeria graminis f. sp. tritici infection tests confirmed additive, race-specific resistance of the two pyramided transgenes in lines Pm17+Pm3b and Pm17+Pm8. Furthermore, pyramided lines showed strong powdery mildew resistance during three field seasons. We conclude that the combination of overexpressed NLR genes from the extended gene pool broadens and diversifies wheat disease resistance.

Breeding for durable resistance against biotrophic fungal pathogens using transgenes from wheat.

Breeding for resistant crops is a sustainable way to control disease and relies on the introduction of novel resistance genes. Here, we tested three strategies on how to use transgenes from wheat to achieve durable resistance against fungal pathogens in the field. First, we tested the highly effective, overexpressed single transgene Pm3e in the background of spring wheat cultivar Bobwhite in a long-term field trial over many years. Together with previous results, this revealed that transgenic wheat line Pm3e#2 conferred complete powdery mildew resistance during a total of nine field seasons without a negative impact on yield. Furthermore, overexpressed Pm3e provided resistance to powdery mildew isolates from our worldwide collection when crossed into the elite wheat cultivar Fiorina. Second, we pyramided the four overexpressed transgenes Pm3a, Pm3b, Pm3d, and Pm3f in the background of cultivar Bobwhite and showed that the pyramided line Pm3a,b,d,f was completely resistant to powdery mildew in five field seasons. Third, we performed field trials with three barley lines expressing adult plant resistance gene Lr34 from wheat during three field seasons. Line GLP8 expressed Lr34 under control of the pathogen-inducible Hv-Ger4c promoter and provided partial barley powdery mildew and leaf rust resistance in the field with small, negative effects on yield components which might need compensatory breeding. Overall, our study demonstrates and discusses three successful strategies for achieving fungal disease resistance of wheat and barley in the field using transgenes from wheat. These strategies might confer long-term resistance if applied in a sustainable way. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01451-2.

Ageing is associated with a reduction in markers of mitochondrial energy metabolism in the human epidermis.

The decline of mitochondrial function throughout the lifespan is directly linked to the development of ageing phenotypes of the skin. Here, we assessed alterations in markers of epidermal mitochondrial energy metabolism as a function of skin age. Human skin samples from distinct anatomical regions were obtained during routine dermatological surgery from 21 young (27.6 ± 1.71 year) and 22 old (76.2 ± 1.73 year) donors. Sections of skin samples were analysed by immunohistochemistry for mitochondrial subunits of each electron transport chain complex (I-V)/oxidative phosphorylation (OXPHOS), as well as proteins serving as a marker of mitochondrial mass (VDAC1) and the regulation of DNA transcription (TFAM). Staining intensities of ATP5F1A (comprising complex V) and TFAM in the epidermis of older subjects were significantly decreased compared with younger donors. Moreover, these effects were independent of UV exposure of the stained skin section. Overall, we demonstrate that ageing is associated with reduced protein levels of complex V of the mitochondrial respiratory chain and TFAM. These alterations may impair essential mitochondrial functions, exacerbating the cutaneous ageing process.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Topical Diacerein Decreases Skin and Splenic CD11c+ Dendritic Cells in Psoriasis.

Psoriasis is an inflammatory skin disease characterized by increased neo-vascularization, keratinocyte hyperproliferation, a pro-inflammatory cytokine milieu and immune cell infiltration. Diacerein is an anti-inflammatory drug, modulating immune cell functions, including expression and production of cytokines, in different inflammatory conditions. Therefore, we hypothesized that topical diacerein has beneficial effects on the course of psoriasis. The current study aimed to evaluate the effect of topical diacerein on imiquimod (IMQ)-induced psoriasis in C57BL/6 mice. Topical diacerein was observed to be safe without any adverse side effects in healthy or psoriatic animals. Our results demonstrated that diacerein significantly alleviated the psoriasiform-like skin inflammation over a 7-day period. Furthermore, diacerein significantly diminished the psoriasis-associated splenomegaly, indicating a systemic effect of the drug. Remarkably, we observed significantly reduced infiltration of CD11c+ dendritic cells (DCs) into the skin and spleen of psoriatic mice with diacerein treatment. As CD11c+ DCs play a pivotal role in psoriasis pathology, we consider diacerein to be a promising novel therapeutic candidate for psoriasis.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Involvement of Neuropeptide Galanin Receptors 2 and 3 in Learning, Memory and Anxiety in Aging Mice.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1-3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12-14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

[Rehab Aftercare 'New Credo' in the Cardiac Follow-Up Rehabilitation]. / Nachsorge 'Neues Credo' in der kardiologischen Anschlussrehabilitation.

PURPOSE: Physical inactivity is considered the most important modifiable risk factor of cardio-vascular diseases. Therefore medical rehabilitation is focused on the improvement of physical activity. To maintain physical activity after rehabilitation aftercare strategies are necessary which help to transfer the skills learned during rehabilitation into daily routine. In this study the aftercare concept "Neues Credo" which has been evaluated several times has been implemented and evaluated into cardiological follow-up rehabilitation. METHODS: Prospective, controlled, multicentre study with 4 cardiological rehabilitation institutions. INCLUSION CRITERIA: rehabilitants with initial diagnosis from the ICD groups I20-25 and I34-43. In the first phase of the study, patients received standard rehabilitation and standard aftercare (control group (KG)). In the second phase, patients received rehabilitation based on the conditions of "Neues Credo" with the focus on increasing physical activity (intervention group (IG)). Data for evaluation were collected by paper-and-pencil questionnaires at 3 points in time. Primary outcome variable: restriction in participation (IMET); secondary outcome variables: depression (CES-D), several scales of subjective health and physical activity. Analysis of variance with repeated measures was used for the evaluation of long-term effects. RESULTS: Complete data could be evaluated from 152 patients of the IG and from 165 patients of the KG. At the end of rehabilitation both IG and KG showed improvements in outcome variables. In the period after rehabilitation patients in the IG improved their physical activity significantly more often than members of the KG (66 vs. 42%, p<0,01), they showed more physical activity than the KG (p=0,040) and they performed endurance sports more often (58 vs. 38%, p<0,01). The primary outcome of participation shows significant improvements in both groups (p<0,01) 12 months after the rehabilitation, difference between groups did not reach statistical significance but indicated a clear tendency in favour of the IG. Similar trends could be found for the secondary outcome-data CONCLUSION: In this study, the "Neues Credo" was applied and evaluated in cardiologic rehabilitation for the first time. Participants reported high practicability and high satisfaction. Health- related outcomes show a trend of positive effects in favour of the IG, but the interaction effects did not reach statistical significance in most cases. Regarding physical activity the intervention group shows clear advantage and will probably benefit from the long-term effects of regular endurance training.

Field grown transgenic Pm3e wheat lines show powdery mildew resistance and no fitness costs associated with high transgene expression.

Pm3 from wheat encodes a nucleotide-binding leucine-rich repeat type of receptor and confers resistance to powdery mildew caused by the fungal pathogen Blumeria graminis f.sp. tritici (Bgt). Each of the 17 functional Pm3 alleles identified so far confers resistance to a distinct spectrum of Bgt isolates. Variant Pm3e has been found in wheat donor line W150 and differs only by two amino acids from the non-functional variant Pm3CS. In order to evaluate the capability of Pm3e to provide powdery mildew field resistance, we generated transgenic Pm3e lines by biolistic transformation of the powdery mildew susceptible spring wheat cultivar Bobwhite. Field trials conducted during four field seasons in Switzerland showed significant and strong powdery mildew resistance of the Pm3e transgenic lines, whereas the corresponding biological sister lines, not containing the transgene, were severely powdery mildew infected. Thus Pm3e alone is responsible for the strong resistance phenotype. The field grown transgenic lines showed high transgene expression and Pm3e protein accumulation with no fitness costs on plant development and yield associated with Pm3e abundance. Line E#1 as well as sister line E#1 showed delayed flowering due to somaclonal variation. The study shows the capability of Pm3e in providing strong powdery mildew field resistance, making its use in wheat breeding programs very promising.

GAL3 receptor knockout mice exhibit an alcohol-preferring phenotype.

Galanin is a neuropeptide which mediates its effects via three G-protein coupled receptors (GAL1-3 ). Administration of a GAL3 antagonist reduces alcohol self-administration in animal models while allelic variation in the GAL3 gene has been associated with an increased risk of alcohol use disorders in diverse human populations. Based on the association of GAL3 with alcoholism, we sought to characterize drug-seeking behavior in GAL3 -deficient mice for the first time. In the two-bottle free choice paradigm, GAL3 -KO mice consistently showed a significantly increased preference for ethanol over water when compared to wildtype littermates. Furthermore, male GAL3 -KO mice displayed significantly increased responding for ethanol under operant conditions. These differences in alcohol seeking behavior in GAL3 -KO mice did not result from altered ethanol metabolism. In contrast to ethanol, GAL3 -KO mice exhibited similar preference for saccharin and sucrose over water, and a similar preference for a high fat diet over a low fat diet as wildtype littermates. No differences in cognitive and locomotor behaviors were observed in GAL3 -KO mice to account for increased alcohol seeking behavior. Overall, these findings suggest genetic ablation of GAL3 in mice increases alcohol consumption.

Evolutionary divergence of the rye Pm17 and Pm8 resistance genes reveals ancient diversity.

KEY MESSAGE: We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes. Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar 'Amigo' and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from 'Petkus' rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

Pathogen-inducible Ta-Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects.

Plant diseases are a serious threat to crop production. The informed use of naturally occurring disease resistance in plant breeding can greatly contribute to sustainably reduce yield losses caused by plant pathogens. The Ta-Lr34res gene encodes an ABC transporter protein and confers partial, durable, and broad spectrum resistance against several fungal pathogens in wheat. Transgenic barley lines expressing Ta-Lr34res showed enhanced resistance against powdery mildew and leaf rust of barley. While Ta-Lr34res is only active at adult stage in wheat, Ta-Lr34res was found to be highly expressed already at the seedling stage in transgenic barley resulting in severe negative effects on growth. Here, we expressed Ta-Lr34res under the control of the pathogen-inducible Hv-Ger4c promoter in barley. Sixteen independent barley transformants showed strong resistance against leaf rust and powdery mildew. Infection assays and growth parameter measurements were performed under standard glasshouse and near-field conditions using a convertible glasshouse. Two Hv-Ger4c::Ta-Lr34res transgenic events were analysed in detail. Plants of one transformation event had similar grain production compared to wild-type under glasshouse and near-field conditions. Our results showed that negative effects caused by constitutive high expression of Ta-Lr34res driven by the endogenous wheat promoter in barley can be eliminated by inducible expression without compromising disease resistance. These data demonstrate that Ta-Lr34res is agronomically useful in barley. We conclude that the generation of a large number of transformants in different barley cultivars followed by early field testing will allow identifying barley lines suitable for breeding.

Pyramiding of transgenic Pm3 alleles in wheat results in improved powdery mildew resistance in the field.

KEY MESSAGE: The combined effects of enhanced total transgene expression level and allele-specificity combination in transgenic allele-pyramided Pm3 wheat lines result in improved powdery mildew field resistance without negative pleiotropic effects. Allelic Pm3 resistance genes of wheat confer race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and encode nucleotide-binding domain, leucine-rich repeat (NLR) receptors. Transgenic wheat lines overexpressing alleles Pm3a, b, c, d, f, and g have previously been generated by transformation of cultivar Bobwhite and tested in field trials, revealing varying degrees of powdery mildew resistance conferred by the transgenes. Here, we tested four transgenic lines each carrying two pyramided Pm3 alleles, which were generated by crossbreeding of lines transformed with single Pm3 alleles. All four allele-pyramided lines showed strongly improved powdery mildew resistance in the field compared to their parental lines. The improved resistance results from the two effects of enhanced total transgene expression levels and allele-specificity combinations. In contrast to leaf segment tests on greenhouse-grown seedlings, no allelic suppression was observed in the field. Plant development and yield scores of the pyramided lines were similar to the mean scores of the corresponding parental lines, and thus, the allele pyramiding did not cause any negative effects. On the contrary, in pyramided line, Pm3b × Pm3f normal plant development was restored compared to the delayed development and reduced seed set of parental line Pm3f. Allele-specific RT qPCR revealed additive transgene expression levels of the two Pm3 alleles in the pyramided lines. A positive correlation between total transgene expression level and powdery mildew field resistance was observed. In summary, allele pyramiding of Pm3 transgenes proved to be successful in enhancing powdery mildew field resistance.

Coronary Collateral Growth Induced by Physical Exercise: Results of the Impact of Intensive Exercise Training on Coronary Collateral Circulation in Patients With Stable Coronary Artery Disease (EXCITE) Trial.

BACKGROUND: A well-developed coronary collateral circulation provides a potential source of blood supply in coronary artery disease. However, the prognostic importance and functional relevance of coronary collaterals is controversial with the association between exercise training and collateral growth still unclear. METHODS AND RESULTS: This prospective, open-label study randomly assigned 60 patients with significant coronary artery disease (fractional flow reserve ≤0.75) to high-intensity exercise (group A, 20 patients) or moderate-intensity exercise (group B, 20 patients) for 4 weeks or to a control group (group C, 20 patients). The primary end point was the change of the coronary collateral flow index (CFI) after 4 weeks. Analysis was based on the intention to treat. After 4 weeks, baseline CFI increased significantly by 39.4% in group A (from 0.142±0.07 at beginning to 0.198±0.09 at 4 weeks) in comparison with 41.3% in group B (from 0.143±0.06 to 0.202±0.09), whereas CFI in the control group remained unchanged (0.7%, from 0.149±0.09 to 0.150±0.08). High-intensity exercise did not lead to a greater CFI than moderate-intensity training. After 4 weeks, exercise capacity, Vo2 peak and ischemic threshold increased significantly in group A and group B in comparison with group C with no difference between group A and group B. CONCLUSIONS: A significant improvement in CFI was demonstrated in response to moderate- and high-intensity exercise performed for 10 hours per week. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01209637.

GAL3 receptor KO mice exhibit an anxiety-like phenotype.

The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems. It is a modulator of various physiological and pathological processes, and it mediates its effects via three G protein-coupled receptors (GAL1-3 receptors). A role for GAL as a modulator of mood and anxiety was suggested, because GAL and its receptors are highly expressed in limbic brain structures of rodents. In recent years, numerous studies of animal models have suggested an involvement of GAL and GAL1 and GAL2 receptors in anxiety- and depression-related behavior. However, to date, there is sparse literature implicating GAL3 receptors in behavioral functions. Therefore, we studied the behavior of GAL3 receptor-deficient (GAL3-KO) mice to elucidate whether GAL3 receptors are involved in mediating behavior-associated actions of GAL. The GAL3-KO mouse line exhibited normal breeding and physical development. In addition to behavioral tests, phenotypic characterization included analysis of hematology, amino acid profiles, metabolism, and sudomotor function. In contrast to WT littermates, male GAL3-KO mice exhibited an anxiety-like phenotype in the elevated plus maze, open field, and light/dark box tests, and they were less socially affiliated than WT animals to a stranger mouse in a social interaction test. In conclusion, our data suggest involvement of GAL3 receptors in GAL-mediated effects on mood, anxiety, and behavior, making it a possible target for alternative treatment strategies for mood disorders.

Galanin 3 receptor-deficient mice show no alteration in the oxazolone-induced contact dermatitis phenotype.

Allergic contact dermatitis (ACD) is an inflammatory skin disease induced by allergen exposure and characterized by erythema, oedema and immune cell infiltration. The sensory peptide galanin (GAL) and its three receptors (GAL1-3 ) are involved in regulating inflammation. As GAL and its receptors are expressed in human and murine skin and GAL expression is increased in oxazolone-induced contact allergy, it could play a role in dermatitis. As GAL reduces neurogenic plasma extravasation in the mouse skin via GAL3 activation, the role of GAL3 in the oxazolone-induced dermatitis model was explored. Following topical challenge with oxazolone, GAL3 gene-deficient mice showed a trend towards reduced ear thickness. Plasma extravasation and neutrophil infiltration increased considerably upon oxazolone challenge in both GAL3 knockout animals and wild-type controls without any observable effect of the gene deletion. We conclude that a lack of GAL3 does not influence oxazolone-induced ACD.

Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3.

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS-containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8-mediated powdery mildew resistance in lines containing Pm8 in a transient single-cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post-translational mechanism is involved in suppression of Pm8. Co-expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co-immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding.

The environment exerts a greater influence than the transgene on the transcriptome of field-grown wheat expressing the Pm3b allele.

Wheat provides 20 % of the calories consumed worldwide. Powdery mildew infections of wheat can result in more than 30 % yield loss but it has been demonstrated that wheat overexpressing Pm3b, an allele of the R gene Pm3, has enhanced resistance against powdery mildew under field conditions. A gene expression profile study using GeneChip Wheat Genome Array and Fluidigm 96.96 Dynamic Arrays was performed to obtain insights into the mode of action of Pm3b and to elucidate the molecular basis of pleiotropic effects observed in three out of four independent transgenic events under field conditions. A cluster analysis of the microarray data and a principal component analysis of the Fluidigm 96.96 Dynamic Arrays data showed that transgenic lines and null segregants grouped together. The microarray analysis of samples from fungicide-treated plants revealed that significantly fewer genes were differentially expressed in Pm3b#1 than in Pm3b#2, which had a pleiotropic phenotype in the field, compared to their null segregants. Together, our data provide evidence that the environment influenced gene expression in the Pm3b lines more than the transgene itself.