Membrane charge dependent states of the β-amyloid fragment Aβ (16-35) with differently charged micelle aggregates

Aβ (16-35) is the hydrophobic central core of β-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, β-amyloid peptides undergo a conformational transition from random coil or α-helical monomers, to highly toxic β-sheet oligomers and aggregate fibrils. The behavior of β-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Aβ (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles “doped” with small amounts of SDS. To analyze the tendency of Aβ (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Aβ (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Aβ (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.     

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G‐protein‐coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl‐Met‐Leu‐Phe) peptide, in a rat basophilic leukaemia RBL‐2H3 cell line stably expressing an HA (haemagglutinin)‐tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist‐induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist‐enhanced cell spreading was inhibited. Analysis of agonist‐stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans‐well migration. The internalization of the formyl‐peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody‐enhanced agonist‐induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant‐induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G‐protein‐coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist‐induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.     

Expression systems and developments in plant-made vaccines

Delivery of vaccines to mucosal surfaces can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle-associated risks. Transgenic plants are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. In the past few years, new techniques (such as chloroplast transformation and food processing) have improved antigen concentration in transgenic plants. In addition, adjuvants and targeting proteins have increased the immunogenicity of mucosally administered plant-made vaccines. These studies have moved plant-made vaccines closer to the development phase.     

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters

BACKGROUND: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes. METHODS: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. RESULTS: Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys.     

Sex allocation in yellow-legged gulls (Larus michahellis) depends on nutritional constraints on production of large last eggs

Male and female offspring can differ in their susceptibility to pre-natal (e.g. egg quality) and post-natal (e.g. sib–sib competition) conditions, and parents can therefore increase their individual fitness by adjusting these maternal effects according to offspring sex. In birds, egg mass and laying/hatching order are the main determinants of offspring viability, but these effects can act differently on each sex. In a previous study, relatively large last-laid (c-)eggs of yellow-legged gulls (Larus michahellis) were more likely to carry a female embryo. This suggests compensatory allocation of maternal resources to daughters from c-eggs, which suffer reduced viability. In the present study, we supplemented yellow-legged gulls with food during the laying period to experimentally test whether their nutritional conditions were responsible for the observed covariation between c-egg sex and mass. As predicted, food supplementation enhanced female c-eggs'' mass more than that of male c-eggs. Thus, this experiment indicates that mothers strategically allocated their resources to c-eggs, possibly in order to compensate for the larger susceptibility of daughters to hatching (and laying) order. The results also suggested that mothers decided on resource allocation depending on the sex of already ovulated c-eggs, rather than ovulating ova of either sex depending on food availability.     

Force spectroscopy of collagen fibers to investigate their mechanical properties and structural organization

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.     

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, Tm, was unchanged, but additional phase transitions appeared above Tm. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small- and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTD-1 above Tm, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption.