Sensory pathways and their modulation in the control of locomotion

Recent experiments have extended our understanding of how sensory information in premotor networks controlling motor output is processed during locomotion, and at what level the efficacy of specific sensory—motor pathways is determined. Phasic presynaptic inhibition of sensory transmission combined with postsynaptic alterations of excitatory and inhibitory synaptic transmission from interneurons of the premotor networks contribute to the modulation of reflex pathways and to the generation of reflex reversal. These mechanisms play an important role in adapting the operation of central networks to external demands and thus help optimize sensory—motor integration.     

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.     

Effect of Isovolemic,Isothermic Hemodialysis on Cerebral Perfusion and Vascular Stiffness Using Contrast Computed Tomography and Pulse Wave Velocity

Background

Patients undergoing hemodialysis treatment have a six-fold increased risk for stroke relative to the general population. However, the effect of hemodialysis on cerebral blood flow is poorly studied and confounding factors like blood pressure and ultrafiltration as well as temperature changes have rarely been accounted for. The aim of our study was to use state-of-the-art technology to evaluate the effect of a single dialysis session on cerebral perfusion as well as on vascular stiffness.

Methods

Chronic hemodialysis patients (7 male/3 female, mean age 58 years) were recruited. Cerebral blood flow and arterial pulse wave velocity were measured before and immediately after a hemodialysis session. To exclude effects of volume changes we kept ultrafiltration to a minimum, allowing no change in body weight. Isothermic conditions were maintained by using the GENIUS single-pass batch-dialysis system with a high-flux polysulfone dialyser. Cerebral blood flow was measured by contrast-enhanced computed tomography. Pulse wave velocity was measured using the SphygmoCor (AtCor Medical, USA) device by a single operator.

Results

This study shows for the first time that isovolemic, isothermic hemodialysis neither affected blood pressure or heart rate, nor total or regional cerebral perfusion. There was also no change in pulse wave velocity.

Conclusions

Mechanisms other than the dialysis procedure itself might be causative for the high incidence of ischemic strokes in this patient population. Moreover, the sole removal of uremic toxins does not lead to short-term effects on vascular stiffness, underlying the importance of volume control in this patient population.     

Zinc uptake and radial transport in roots of Arabidopsis thaliana: a modelling approach to understand accumulation

Background and Aims

Zinc uptake in roots is believed to be mediated by ZIP (ZRT-, IRT-like proteins) transporters. Once inside the symplast, zinc is transported to the pericycle, where it exits by means of HMA (heavy metal ATPase) transporters. The combination of symplastic transport and spatial separation of influx and efflux produces a pattern in which zinc accumulates in the pericycle. Here, mathematical modelling was employed to study the importance of ZIP regulation, HMA abundance and symplastic transport in creation of the radial pattern of zinc in primary roots of Arabidopsis thaliana.

Methods

A comprehensive one-dimensional dynamic model of radial zinc transport in roots was developed and used to conduct simulations. The model accounts for the structure of the root consisting of symplast and apoplast and includes effects of water flow, diffusion and cross-membrane transport via transporters. It also incorporates the radial geometry and varying porosity of root tissues, as well as regulation of ZIP transporters.

Key Results

Steady-state patterns were calculated for various zinc concentrations in the medium, water influx and HMA abundance. The experimentally observed zinc gradient was reproduced very well. An increase of HMA or decrease in water influx led to loss of the gradient. The dynamic behaviour for a change in medium concentration and water influx was also simulated showing short adaptation times in the range of seconds to minutes. Slowing down regulation led to oscillations in expression levels, suggesting the need for rapid regulation and existence of buffering agents.

Conclusions

The model captures the experimental findings very well and confirms the hypothesis that low abundance of HMA4 produces a radial gradient in zinc concentration. Surprisingly, transpiration was found also to be a key parameter. The model suggests that ZIP regulation takes place on a comparable timescale as symplastic transport.     

Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.     

Biophysics and bioinformatics reveal structural differences of the two peripheral stalk subunits in chloroplast ATP synthase

ATP synthases convert an electrochemical proton gradient into rotational movement to produce the ubiquitous energy currency adenosine triphosphate. Tension generated by the rotational torque is compensated by the stator. For this task, a peripheral stalk flexibly fixes the hydrophilic catalytic part F1 to the membrane integral proton conducting part F(O) of the ATP synthase. While in eubacteria a homodimer of b subunits forms the peripheral stalk, plant chloroplasts and cyanobacteria possess a heterodimer of subunits I and II. To better understand the functional and structural consequences of this unique feature of photosynthetic ATP synthases, a procedure was developed to purify subunit I from spinach chloroplasts. The secondary structure of subunit I, which is not homologous to bacterial b subunits, was compared to heterologously expressed subunit II using CD and FTIR spectroscopy. The content of alpha-helix was determined by CD spectroscopy to 67% for subunit I and 41% for subunit II. In addition, bioinformatics was applied to predict the secondary structure of the two subunits and the location of the putative coiled-coil dimerization regions. Three helical domains were predicted for subunit I and only two uninterrupted domains for the shorter subunit II. The predicted length of coiled-coil regions varied between different species and between subunits I and II.     

Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase

The current study examined the aromatic degradation and central metabolism in Corynebacterium glutamicum by proteomic and molecular methods. Comparative analysis of proteomes from cells grown on gentisate and on glucose revealed that 30% of the proteins of which their abundance changed were involved in aromatic degradation and central carbon metabolism. Similar results were obtained from cells grown on benzoate, 4-cresol, phenol, and resorcinol. Results from these experiments revealed that (i) enzymes involved in degradation of benzoate, 4-cresol, gentisate, phenol, and resorcinol were specifically synthesized and (ii) that the abundance of enzymes involved in central carbon metabolism of glycolysis/gluconeogenesis, pentose phosphate pathway, and TCA cycles were significantly changed on various aromatic compounds. Significantly, three novel proteins, NCgl0524, NCgl0525, and NCgl0527, were identified on 4-cresol. The genes encoding NCgl0525 and NCgl0527 were confirmed to be necessary for assimilation of 4-cresol with C. glutamicum. The abundance of fructose-1,6-bisphosphatase (Fbp) was universally increased on all the tested aromatic compounds. This Fbp gene was disrupted and the mutant WT(Deltafbp) lost the ability to grow on aromatic compounds. Genetic complementation by the Fbp gene restored this ability. We concluded that gluconeogenesis is a necessary process for C. glutamicum growing on various aromatic compounds.