Single particle analysis of tau oligomer formation induced by metal ions and organic solvents

Pathological aggregates of tau protein are found in several neurodegenerative diseases termed ‘tauopathies’. Increasing evidence indicates that tau oligomer species rather than the large amyloid cytoplasmic inclusions relevant for histopathological diagnosis might be crucial for cellular damage and neurodegeneration. Trivalent metal ions and polyanionic structures like heparin or arachidonic acid have been shown to induce tau aggregation. However, little is known about early processes of tau aggregation. In this study, we applied fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT) to investigate oligomer formation of tau protein at nanomolar protein concentrations at the single-particle level. Our results indicate that the formation of distinct tau oligomers is induced by the trivalent metal ions Fe³⁺ and Al³⁺ and by organic solvents like DMSO, respectively. In contrast, bivalent metal ions (Cu²⁺, Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺) had no effect. While DMSO-induced small tau oligomers are relatively stable in solution, dynamic remodeling can be initiated by non-ionic detergents. Moreover Al³⁺ induces rapid formation of a different oligomer species of larger size. Our results provide further insights into early tau oligomerization and aggregation dynamics.     

NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin

The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.     

Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies

This communication presents a novel experimental model for Alzheimer studies, where connected primary neurons were set into subtend, co-pathological states. Cortical neurons were cultured in two separated cell compartments in a microfluidic device. A neurite network was generated in a main channel through the neurite outgrowth from both cell compartments. A gradient of okadaic acid (OA) is generated over this neurite network by perfusion. OA is a phosphatase inhibitor that induces hyperphosphorylation of Tau proteins, a major hallmark in Alzheimer disease. The local OA treatment resulted in a connected "diseased" and "healthy" cell population. Anti-phosphorylated tau (Ser262) staining confirmed different states of phosphorylated Tau proteins, and synapthophysin staining the connection of "healthy" and "diseased" cells. Here, we present a novel in vitro model that opens the possibility to study cellular and molecular propagation mechanisms in neurodegeneration, in Tauopathies (as e.g., in Alzheimer), as well as simultaneous drug effects on connected healthy and diseased cell populations.     

Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation

Mesenchymal stem cells (MSCs) possess immunosuppressive properties. But also fully differentiated human renal tubular epithelial cells (RTECs) are able to modulate T-cell proliferation in vitro. In this study we compared two MSC populations, human adipose derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs), and RTECs regarding their potential to inhibit monocyte-derived dendritic cell (DC) differentiation and maturation in indirect co-culture.In the presence of hAMSCs and RTECs, monocytes stimulated to undergo DC differentiation were inhibited to acquire surface phenotype of immature and mature DCs. In contrast, ASCs showed only limited suppressive capacity. Secretion of IL-12p70 was suppressed in hAMSC co-cultures and high IL-10 levels were detected in all co-cultures. Prostaglandin E₂ was found in ASC and hAMSC co-cultures, whereas soluble human leukocyte antigen-G was highly elevated only in RTEC co-cultures. Thus, inhibition of DC generation by MSCs and RTECs might be mediated by different soluble factors.     

The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs

In order to determine the effect of pectin on fermentation parameters in the faeces and caecal digesta of weaned pigs 18 castrated male crossbred pigs with an average body weight of 8?kg were fitted with T-cannulas at the caecum. The animals were randomly distributed into three groups and fed with diets supplemented with 0, 5 and 10% pectin. Faeces were collected over a period of 3 days. Thereafter the diets were withdrawn for 24?h followed by ad libitum feeding to enhance the feed intake. Caecal chyme was collected 0, 8 and 24?h postprandial. In the faeces the addition of 5% pectin to the diet lowered the content of dry matter and lactic acid. The pH and the digestibility of pectins, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. When the diets were withdrawn and fed ad libitum 24?h later, a decline of the pH and an increased concentration of lactate in the caecal chyme could be observed in all groups up to 8?h after feeding. With an interval of 8 to 24?h after feeding, a further decline in pH and a rise of lactate only occurred when the diet was not supplemented with pectin. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine.     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

Novel Bioimaging Techniques of Metals by Laser Ablation Inductively Coupled Plasma Mass Spectrometry for Diagnosis Of Fibrotic and Cirrhotic Liver Disorders

Background and Aims

Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.

Methods

In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.

Results

Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.

Conclusions

Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.     

Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.     

The Hedgehog Receptor Patched1 in T Cells Is Dispensable for Adaptive Immunity in Mice

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and T_reg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.     

Proteomic and Immunochemical Characterization of Glutathione Transferase as a New Allergen of the Nematode Ascaris lumbricoides

Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules.Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.