Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

Evaluation of the leptin receptor in human spermatozoa

Background  

Leptin, a 167 amino acid peptide hormone, profoundly effects reproduction exerting its biological effects via interaction with the leptin receptor (ObR) which is widely expressed on peripheral tissues. In this study, we have attempted to assess leptin receptor expression in the spermatozoa of fertile males and those diagnosed with male factor infertility; both at the mRNA or protein levels.     

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (Z_Aβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z_Aβ3 in the brains of Drosophila melanogaster expressing either Aβ₄₂ or the aggressive familial associated E22G variant of Aβ₄₂ abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that Z_Aβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.     

Angiostatin effects on endothelial cells mediated by ceramide and RhoA

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean

Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world''s largest continuous rhodolith bed (of ∼21 000 km²) and has one of the largest marine CaCO₃ deposits (producing 25 megatons of CaCO₃ per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 μmol carbon m⁻²s⁻¹), allowing the entire Abrolhos rhodolith bed to produce 5.65 × 10⁵ tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean.     

Stimuli-Responsive magnetic nanoparticles for monoclonal antibody purification

Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.     

Molecular evidence and clinical importance of β‐arrestins expression in patients with acromegaly

β‐arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β‐arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β‐arrestins with response to first‐generation SRL and invasiveness in somatotropinomas. β‐arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real‐time RT‐PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF‐I levels, was assessed in 40 patients. The Knosp‐Steiner criteria were used to define invasiveness. Median β‐arrestin 1, β‐arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156 ; and 3989, respectively. There was a positive correlation between β‐arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β‐arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β‐arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β‐arrestins and sst2, our data clearly indicated that no association existed between β‐arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β‐arrestins have a role in the response to treatment with SRL in acromegaly.