Phosphorylation and membrane dissociation of the ARF exchange factor GBF1 in mitosis

Secretory protein trafficking is arrested and the Golgi apparatus fragmented when mammalian cells enter mitosis. These changes are thought to facilitate cell cycle progression and Golgi inheritance, and are brought about through the actions of mitotically active protein kinases. To better understand how the Golgi apparatus undergoes mitotic fragmentation we have sought to identify novel Golgi targets for mitotic kinases. We report here the identification of the ARF exchange factor GBF1 as a Golgi phosphoprotein. GBF1 is phosphorylated by CDK1-cyclin B in mitosis, which results in its dissociation from Golgi membranes. Consistent with a reduced level of GBF1 activity at the Golgi membrane there is a reduction in levels of membrane-associated GTP-bound ARF in mitotic cells. Despite the reduced levels of membrane bound GBF1 and ARF, COPI binding to the Golgi membrane appears unaffected in mitotic cells. Surprisingly, this pool of COPI is dependent upon GBF1 for its recruitment to the membrane, suggesting a low level of GBF1 activity persists in mitosis. We propose that the phosphorylation and membrane dissociation of GBF1 and the consequent reduction in ARF-GTP levels in mitosis are important for changes in Golgi dynamics and possibly other mitotic events mediated through effectors other than the COPI vesicle coat.

Evidence for dissociation between the perceptual and visuomotor systems in humans

When a visual stimulus is continuously moved behind a small stationary window, the window appears displaced in the direction of motion of the stimulus. In this study we showed that the magnitude of this illusion is dependent on (i) whether a perceptual or visuomotor task is used for judging the location of the window, (ii) the directional signature of the stimulus, and (iii) whether or not there is a significant delay between the end of the visual presentation and the initiation of the localization measure. Our stimulus was a drifting sinusoidal grating windowed in space by a stationary, two-dimensional, Gaussian envelope (σ=1 cycle of sinusoid). Localization measures were made following either a short (200 ms) or long (4.2 s) post-stimulus delay. The visuomotor localization error was up to three times greater than the perceptual error for a short delay. However, the visuomotor and perceptual localization measures were similar for a long delay. Our results provide evidence in support of the hypothesis that separate cortical pathways exist for visual perception and visually guided action and that delayed actions rely on stored perceptual information.

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

A developmental dissociation of view-dependent and view-invariant object recognition in adolescence

Spatial generalization skills in school children aged 8-16 were studied with regard to unfamiliar objects that had been previously learned in a cross-modal priming and learning paradigm. We observed a developmental dissociation with younger children recognizing objects only from previously learnt perspectives whereas older children generalized acquired object knowledge to new viewpoints as well. Haptic and - to a lesser extent - visual priming improved spatial generalization in all but the youngest children. The data supports the idea of dissociable, view-dependent and view-invariant object representations with different developmental trajectories that are subject to modulatory effects of priming. Late-developing areas in the parietal or the prefrontal cortex may account for the retarded onset of view-invariant object recognition. © 2006 Elsevier B.V. All rights reserved.

Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Fractionating the unitary notion of dissociation:disembodied but not embodied dissociative experiences are associated with exocentric perspective-taking

It has been argued that hallucinations which appear to involve shifts in egocentric perspective (e.g., the out-of-body experience, OBE) reflect specific biases in exocentric perspective-taking processes. Via a newly devised perspective-taking task, we examined whether such biases in perspective-taking were present in relation to specific dissociative anomalous body experiences (ABE) - namely the OBE. Participants also completed the Cambridge Depersonalization Scale (CDS; Sierra and Berrios, 2000) which provided measures of additional embodied ABE (unreality of self) and measures of derealization (unreality of surroundings). There were no reliable differences in the level of ABE, emotional numbing, and anomalies in sensory recall reported between the OBE and control group as measured by the corresponding CDS subscales. In contrast, the OBE group did provide significantly elevated measures of derealization ("alienation from surroundings" CDS subscale) relative to the control group. At the same time we also found that the OBE group was significantly more efficient at completing all aspects of the perspective-taking task relative to controls. Collectively, the current findings support fractionating the typically unitary notion of dissociation by proposing a distinction between embodied dissociative experiences and disembodied dissociative experiences - with only the latter being associated with exocentric perspective-taking mechanisms. Our findings - obtained with an ecologically valid task and a homogeneous OBE group - also call for a re-evaluation of the relationship between OBEs and perspective-taking in terms of facilitated disembodied experiences. © 2013 Braithwaite, James, Dewe, Takahashi, Medford and Kessler.

Effects of short-term detraining on postprandial metabolism, endothelial function, and inflammation in endurance-trained men:dissociation between changes in triglyceride metabolism and endothelial function

Endurance-trained athletes experience a low level of postprandial lipaemia, but this rapidly increases with detraining. We sought to determine whether detraining-induced changes to postprandial metabolism influenced endothelial function and inflammation. Eight endurance-trained men each undertook two oral fat tolerance tests [blood taken fasted and for 6 h following a high-fat test meal (80 g fat, 80 g carbohydrate)]: one during a period of their normal training (trained) and one after 1 wk of no exercise (detrained). Endothelial function in the cutaneous microcirculation was assessed using laser Doppler imaging with iontophoresis in the fasted state and 4 h postprandially during each test. Fasting plasma triglyceride (TG) concentrations increased by 35% with detraining (P = 0.002), as did postprandial plasma (by 53%, P = 0.002), chylomicron (by 68%, P = 0.02) and very low-density lipoprotein (by 51%, P = 0.005) TG concentrations. Endothelial function decreased postprandially in both the trained (by 17%, P = 0.03) and detrained (by 22%, P = 0.03) conditions but did not differ significantly between the trained and detrained conditions in either the fasted or the postprandial states. These results suggest that, although fat ingestion induces endothelial dysfunction, interventions that alter postprandial TG metabolism will not necessarily concomitantly influence endothelial function.

Developmental dissociation of analytical and holistic object recognition in adolescence

Previous research (e.g., Jüttner et al, 2013, Developmental Psychology, 49, 161-176) has shown that object recognition may develop well into late childhood and adolescence. The present study extends that research and reveals novel di erences in holistic and analytic recognition performance in 7-11 year olds compared to that seen in adults. We interpret our data within Hummel’s hybrid model of object recognition (Hummel, 2001, Visual Cognition, 8, 489-517) that proposes two parallel routes for recognition (analytic vs. holistic) modulated by attention. Using a repetition-priming paradigm, we found in Experiment 1 that children showed no holistic priming, but only analytic priming. Given that holistic priming might be thought to be more ‘primitive’, we confirmed in Experiment 2 that our surprising finding was not because children’s analytic recognition was merely a result of name repetition. Our results suggest a developmental primacy of analytic object recognition. By contrast, holistic object recognition skills appear to emerge with a much more protracted trajectory extending into late adolescence

Dissociation of detection and evaluation of facial expressions in adolescence

Holistic face perception, i.e. the mandatory integration of featural information across the face, hasbeen considered to play a key role when recognizing emotional face expressions (e.g., Tanaka et al.,2002). However, despite their early onset holistic processing skills continue to improvethroughout adolescence (e.g., Schwarzer et al., 2010) and therefore might modulate theevaluation of facial expressions. We tested this hypothesis using an attentional blink (AB)paradigm to compare the impact of happy, fearful and neutral faces in adolescents (10–13 years)and adults on subsequently presented neutral target stimuli (animals, plants and objects) in a rapidserial visual presentation stream. Adolescents and adults were found to be equally reliable whenreporting the emotional expression of the face stimuli. However, the detection of emotional butnot neutral faces imposed a significantly stronger AB effect on the detection of the neutral targetsin adults compared to adolescents. In a control experiment we confirmed that adolescents ratedemotional faces lower in terms of valence and arousal than adults. The results suggest a protracteddevelopment of the ability to evaluate facial expressions that might be attributed to the latematuration of holistic processing skills.