Vibratory Communication in the Jumping Spider Phidippus clarus: Substrate‐borne Courtship Signals are Important for Male Mating Success

Recently, work has shown that multimodal communication is common throughout the animal kingdom but the function of multimodal signals is still poorly understood. Phidippus clarus are jumping spiders in which males produce multimodal (visual and vibrational) signals in both male–male (aggressive) and male–female (courtship) contexts. The P. clarus mating system is complex, with sex ratios and the level of male competition changing over the course of the breeding season. Vibrational signal components have been shown to function in male aggressive contests but their role in courtship has not been investigated. Here, we performed an experiment to test the role of vibrational signaling in courtship by observing mating success for males that were experimentally muted. We show that vibratory courtship signals, and in particular signaling rate, is an important component of mating success and potentially a target of female choice. While the ability to produce vibratory signals significantly increased mating success, some muted males were still able to successfully mate. In these trials, signaling rate also predicted mating success suggesting that redundant signal components may compensate for errors and perturbations in signal transmission or that vibratory signals function to enhance the efficacy of visual signals.     

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimer's disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[¹⁸F]FMeNER-D₂. (S,S)-[¹⁸F]FMeNER-D₂ appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[¹⁸F]FMeNER-D₂ in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[¹⁸F]FMeNER-D₂ as a molecular imaging biomarker in AD.     

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.     

Comparative aspects of in vitro proliferation of human and porcine lymphocytes exposed to mycotoxins

Mycotoxins are fungal secondary metabolites that elicit a wide spectrum of toxicological effects, including the alteration of normal immune function. In the present study we investigated the independent effect of four mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1), deoxynivalenol (DON) and nivalenol (NIV), on lymphocyte proliferation using human and porcine lymphocytes. Human and porcine peripheral blood mononuclear cells and porcine splenocytes were cultured with increasing concentrations of mycotoxins for 72 hours and labelled in the last 24 hours with [methyl-3H]-thymidine. The results showed that increased concentrations of AFB1, DON and NIV affected the [methyl-3H]-thymidine cellular proliferation following mitogen stimulation in both species and cell types. Lower concentrations of mycotoxins enhanced cellular proliferation, which was more pronounced in human than in porcine cells, while higher concentrations caused a dose-dependent decrease. DON and NIV were the most potent mycotoxin in both species and both cell types. Based on the results of this in vitro study, high correlations were found between proliferation of human and porcine lymphocytes after mycotoxin exposure, especially for DON and NIV.     

Genetic mouse models of Huntington’s disease: focus on electrophysiological mechanisms

The discovery of the HD (Huntington’s disease) gene in 1993 led to the creation of genetic mouse models of the disease and opened the doors for mechanistic studies. In particular, the early changes and progression of the disease could be followed and examined systematically. The present review focuses on the contribution of these genetic mouse models to the understanding of functional changes in neurons as the HD phenotype progresses, and concentrates on two brain areas: the striatum, the site of most conspicuous pathology in HD, and the cortex, a site that is becoming increasingly important in understanding the widespread behavioural abnormalities. Mounting evidence points to synaptic abnormalities in communication between the cortex and striatum and cell–cell interactions as major determinants of HD symptoms, even in the absence of severe neuronal degeneration and death.     

Regulation of the gibberellin pathway by auxin and DELLA proteins

The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel’s LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA₂₀ showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.     

Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.The structure of many proteins can be altered by post-translational modifications (1). Although the impact of post-translational modifications (PTMs)¹ on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2), it is less defined for other PTMs and in many cases is protein-dependent. Because there are many important downstream effects of PTMs, including changes in protein localization, protein and cellular diversification, protein functionality, protein stability, protein life cycle, and so forth, understanding how PTMs alter protein structure for as many proteins as possible in a timely manner is a highly desirable goal. Furthermore, in an age where recombinant proteins are being used to treat disease, it becomes ever more important to understand how particular modifications may alter the structure and eventually the function of therapeutic proteins. To realize these goals, methods that permit access to conformational information for modified forms of therapeutic proteins must be developed and refined. In this report, we will illustrate how MS can contribute to structural proteomics by describing our recent work with a recombinant monoclonal antibody (an IgG1), which represents an important class of therapeutic proteins.Many biopharmaceutical companies are pursuing antibody drugs (3). In particular, the IgG1 subclass of antibodies has evolved into a commonly used therapeutic option for the treatment of a wide range of diseases. IgG1s consist of a dimer of identical heavy chains and light chains that fold to form (from N to C terminus) the variable, CL, CH1, CH2, and CH3 domains (as an example, see Ref. 4). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5). The variable, CL, and CH1 domains are collectively referred to as the Fab (fragment antigen binding) portion of IgG1, which is responsible for recognizing a specific antigen. The CH2 and CH3 domains together are referred to as the Fc (fragment crystallizable) portion, which carries out effector functions such as binding to Fcγ receptors. These effector functions are essential to many therapeutic antibodies, especially when antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity are involved in the mechanisms of action (6).As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood at a functional level. In the case of IgG1s, with over 1300 amino acid residues and a molecular mass approaching 150 kDa, a large array of PTMs can be incorporated both in vivo (during cellular synthesis) and in vitro (as a result of handling and processing steps that occur during purification, vialing, and storage). Commonly monitored PTMs on IgG1s include methionine oxidation, asparagine and glutamine deamidation, N-terminal acetylation or cyclization, glycation of lysine, and variable glycosylation (8). Some of these modifications affect only a small percentage of the protein product, and their presence may not change overall outcome. Others, however, can have significant impact on the structure, function, and biological activities of a protein that can involve self-association as well as interactions with other proteins (9). The same PTMs can affect different IgG1 molecules in different ways or have no effect(s) at all. Therefore assessing the presence of PTMs, determining the relative level of the modifications, and understanding the structural effects of PTMs are all important during development of protein biopharmaceuticals.Two commonly studied IgG1 modifications are methionine oxidation and glycosylation, each of which has been shown to affect biological function (6, 10). Methionine oxidation has been implicated in protein stability (inducing aggregation), and increased oxidation levels have been shown to provoke an immunogenic response (11–13). Elevated levels of methionine oxidation in an IgG1 were shown to impact neonatal Fc receptor (FcRn) and protein A binding (10). Variable glycosylation (i.e. different levels of sialic acid, galactose, fucose, or high mannose structures) is known to influence thermal stability and effector functions (14–16). Previous studies have shown that removal of fucose from the glycan present on the Fc portion of an IgG1 can greatly enhance Fc binding to FcγRIIIa, but removal of the entire glycan nearly abolishes FcγRIIIa binding (17). As oxidation and changes to the glycan are both common IgG1 modifications, we were interested in determining the conformational effects of oxidation, afucosylation, and galactosylation and correlating any conformational changes that were observed with changes of FcγRIIIa binding activity.Conformational analysis of large proteins like antibodies, however, is not trivial. Traditional biophysical techniques such as circular dichroism, DSC, and fluorescence provide useful information, but these techniques look at the entire protein and provide only a global view (18). NMR and x-ray crystallography can both provide high resolution structural analysis, but each is faced with limitations that often make the study of an intact IgG1 difficult or nearly impossible (19–21). Recently we described how hydrogen/deuterium exchange (H/DX) MS could be used to study the conformation and conformational dynamics of an intact IgG1 with resolution down to stretches of several amino acid residues (22). For the present work, we used H/DX MS to study the impact of galactosylation, oxidation, and afucosylation on the conformation and dynamics of an intact IgG1. We also studied the complex of IgG1 and FcγRIIIa to map the points of interaction and probe any changes in the dynamics of the IgG1 as a result of FcγRIIIa interaction. Finally, we correlated the functional activity of all the proteins that were studied by H/DX MS with the observed conformational disturbance(s). Such correlations are important to connect structure with function and to understand whether a particular PTM is something that may affect the therapeutic value of a recombinant protein.     

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.     

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin‐8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.     

The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalators

Fifteen platinum(II)-based metallointercalators have been synthesised that utilise substituted 1,10-phenanthroline (phen) ligands, including 5-chloro-1,10-phenanthroline (5-Cl-phen), 5-methyl-1,10-phenanthroline (5-CH3-phen), 5-amino-1,10-phenanthroline (5-NH2-phen), 5-nitro-1,10-phenanthroline (5-NO2-phen) and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), and achiral ethylenediamine (en) and the chiral ancillary ligands 1S,2S-diaminocyclohexane (S,S-dach) and 1R,2R-diaminocyclohexane (R,R-dach). Their cytotoxicity in the L1210 murine leukaemia cell line was determined using growth inhibition assays. The most cytotoxic metal complexes are those that contain S,S-dach ancillary ligands and 5-CH3-phen intercalating ligands. One metallointercalator [Pt(5-CH3-phen)(S,S-dach)]Cl2 (5MESS), displays a 5-10-fold increase in cytotoxicity compared to the clinical agent cisplatin. From DNA binding experiments there appears to be no significant difference between any of the metal complexes, indicating that neither DNA binding affinity nor the mode of binding/DNA adduct formed is the sole determinant of the cytotoxicity of this family of platinum(II)-based metallointercalators.