Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Pacemaking by HCN channels requires interaction with phosphoinositides

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels mediate the depolarizing cation current (termed I(h) or I(f)) that initiates spontaneous rhythmic activity in heart and brain. This function critically depends on the reliable opening of HCN channels in the subthreshold voltage-range. Here we show that activation of HCN channels at physiologically relevant voltages requires interaction with phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) acts as a ligand that allosterically opens HCN channels by shifting voltage-dependent channel activation approximately 20 mV toward depolarized potentials. Allosteric gating by PIP(2) occurs in all HCN subtypes and is independent of the action of cyclic nucleotides. In CNS neurons and cardiomyocytes, enzymatic degradation of phospholipids results in reduced channel activation and slowing of the spontaneous firing rate. These results demonstrate that gating by phospholipids is essential for the pacemaking activity of HCN channels in cardiac and neuronal rhythmogenesis.     

G protein-coupled receptor 83 overexpression in naive CD4+CD25- T cells leads to the induction of Foxp3+ regulatory T cells in vivo

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3-CD4+CD25- T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25- or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25- T cells. Transduction of naive CD4+CD25- T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.     

Self-limitation of Th1-mediated inflammation by IFN-gamma

IFN-gamma is an effector cytokine of cell-mediated immunity that plays an essential role in both innate and adaptive phases of an immune response. Interestingly, in several Th1-dependent autoimmune models, lack of IFN-gamma is associated with an acceleration of disease. To distinguish the influence of IFN-gamma on the polarization of naive precursors from the influence on effector cells, we used an adoptive transfer model of differentiated Ag-specific Th1 cells. In this study, IFN-gamma displayed a dual function in a Th1-dependent immune reaction. In the early phase, IFN-gamma accelerated the inflammation, whereas in the late phase it mediated the process of self-limitation. We demonstrated that IFN-gamma limits the number of Th1 effector cells after Ag challenge. Studies using IFN-gammaR-/- mice as recipients showed that IFN-gamma acts indirectly via host cells to regulate the pool size of Th1 cells. NO was a downstream effector molecule. Transfer experiments of Th1 cells into IFN-gamma-/- mice revealed that Th1 cells control both themselves and the corresponding inflammation by the release of IFN-gamma. Thus, the proinflammatory cytokine IFN-gamma can act as a negative feedback regulator to control Th1-mediated immune responses.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Tree species composition and diversity gradients in white-water forests across the Amazon Basin

Aim Attention has increasingly been focused on the floristic variation within forests of the Amazon Basin. Variations in species composition and diversity are poorly understood, especially in Amazonian floodplain forests. We investigated tree species composition, richness and α diversity in the Amazonian white‐water (várzea) forest, looking particularly at: (1) the flood‐level gradient, (2) the successional stage (stand age), and (3) the geographical location of the forests. Location Eastern Amazonia, central Amazonia, equatorial western Amazonia and the southern part of western Amazonia. Methods The data originate from 16 permanent várzea forest plots in the central and western Brazilian Amazon and in the northern Bolivian Amazon. In addition, revised species lists of 28 várzea forest inventories from across the Amazon Basin were used. Most important families and species were determined using importance values. Floristic similarity between plots was calculated to detect similarity variations between forest types and over geographical distances. To check for spatial diversity gradients, α diversity (Fisher) of the plots was correlated with stand age, longitudinal and latitudinal plot location, and flood‐level gradient. Results More than 900 flood‐tolerant tree species were recorded, which indicates that Amazonian várzea forests are the most species‐rich floodplain forests worldwide. The most important plant families recorded also dominate most Neotropical upland forests, and c. 31% of the tree species listed also occur in the uplands. Species distribution and diversity varied: (1) on the flood‐level gradient, with a distinct separation between low‐várzea forests and high‐várzea forests, (2) in relation to natural forest succession, with species‐poor forests in early stages of succession and species‐rich forests in later stages, and (3) as a function of geographical distance between sites, indicating an increasing α diversity from eastern to western Amazonia, and simultaneously from the southern part of western Amazonia to equatorial western Amazonia. Main conclusions The east‐to‐west gradient of increasing species diversity in várzea forests reflects the diversity patterns also described for Amazonian terra firme. Despite the fine‐scale geomorphological heterogeneity of the floodplains, and despite high disturbance of the different forest types by sedimentation and erosion, várzea forests are dominated by a high proportion of generalistic, widely distributed tree species. In contrast to high‐várzea forests, where floristic dissimilarity increases significantly with increasing distance between the sites, low‐várzea forests can exhibit high floristic similarity over large geographical distances. The high várzea may be an important transitional zone for lateral immigration of terra firme species to the floodplains, thus contributing to comparatively high species richness. However, long‐distance dispersal of many low‐várzea trees contributes to comparatively low species richness in highly flooded low várzea.     

Volatiles modulate the development of plant pathogenic rust fungi

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.     

Hybrid maize breeding with doubled haploids: I. One-stage versus two-stage selection for testcross performance

Optimum allocation of resources is of fundamental importance for the efficiency of breeding programs. The objectives of our study were to (1) determine the optimum allocation for the number of lines and test locations in hybrid maize breeding with doubled haploids (DHs) regarding two optimization criteria, the selection gain ΔG _k and the probability P _k of identifying superior genotypes, (2) compare both optimization criteria including their standard deviations (SDs), and (3) investigate the influence of production costs of DHs on the optimum allocation. For different budgets, number of finally selected lines, ratios of variance components, and production costs of DHs, the optimum allocation of test resources under one- and two-stage selection for testcross performance with a given tester was determined by using Monte Carlo simulations. In one-stage selection, lines are tested in field trials in a single year. In two-stage selection, optimum allocation of resources involves evaluation of (1) a large number of lines in a small number of test locations in the first year and (2) a small number of the selected superior lines in a large number of test locations in the second year, thereby maximizing both optimization criteria. Furthermore, to have a realistic chance of identifying a superior genotype, the probability P _k of identifying superior genotypes should be greater than 75%. For budgets between 200 and 5,000 field plot equivalents, P _k > 75% was reached only for genotypes belonging to the best 5% of the population. As the optimum allocation for P _k (5%) was similar to that for ΔG _k , the choice of the optimization criterion was not crucial. The production costs of DHs had only a minor effect on the optimum number of locations and on values of the optimization criteria. C. Friedrich H. Longin and H. Friedrich Utz contributed equally to this work.     

A new test for family-based association mapping with inbred lines from plant breeding programs

Association mapping holds great promise for the detection of quantitative trait loci (QTL) in plant breeding populations. The main objectives of this study were to (1) adapt the quantitative pedigree disequilibrium test to typical pedigrees of inbred lines produced in plant breeding programs, (2) compare the newly developed quantitative inbred pedigree disequilibrium test (QIPDT) with the commonly employed logistic regression ratio test (LRRT), with respect to the power and type I error rate of QTL detection, and (3) demonstrate the use of the QIPDT by applying it to flowering data of European elite maize inbreds. QIPDT and LRRT were compared based on computer simulations modeling 55 years of hybrid maize breeding in Central Europe. Furthermore, we applied QIPDT to a cross-section of 49 European elite maize inbred lines genotyped with 722 amplified fragment length polymorphism markers and phenotyped in four environments for days to anthesis. Compared to LRRT, the power to detect QTL was higher with QIPDT when using data collected routinely in plant breeding programs. Application of QIPDT to the 49 European maize inbreds resulted in a significant (P < 0.05) association located at a position for which a consensus QTL was detected in a previous study. The results of our study suggested that QIPDT is a promising QTL detection method for data collected routinely in plant breeding programs.