Residual motion of hemoglobin-bound spin labels and protein dynamics: viscosity dependence of the rotational correlation times

The residual motion of spin labels bound to cysteine 93 and to lysines of methemoglobin has been studied by electron paramagnetic resonance spectroscopy. To separate the influences of the solvent and the protein environment of the label fluctuations, the correlation times, , were analyzed as a function of temperature for fixed solvent viscosities, . Results show that over a wide range of viscosity the dependence of on may be empirically described by a power law ^k . The exponent k depends strongly on the location of the label on the protein surface. If one regards the spin labels as artificial amino acid side chains, characteristic values of correlation times and amplitudes of the rotational motion at the surface can be given. For =1 cP and T=297 K the correlation time of the labels bound to lysines is found to be =9 · 10^–10 s and the rotational diffusion is nearly isotropic. The spin label bound to cysteine 93 occupies a protein pocket, its rotational motion is therefore restricted. The correlation time of the label motion within a limited motion cone of semi angle =30° ± 3° is found to be =1.3 · 10^–9 s for =1 cP and T=297 K.     

Zechstein 2 carbonate platform subfacies and grain-type distribution (Upper Permian,Northwest Germany)

Summary The Upper Permian Zechstein 2 Carbonate (Stassfurt Carbonate, or Ca2) platform facies of Northwest Germany can be subdivided into twelve subfacies types using slabbed cores from fifteen representative wells. Thin section and scanning microscopic analysis further provide subfacies-specific characteristics, based on distribution, size, shape, and spatial arrangement of the grains contained in the different subfacies types. Thirteen grain types can be distinguished within the different subfacies types on the Ca2-platform: 1) one type of oncoid, 2) one type of grapestone, 3) three types of peloids, 4) four types of ooids and 5) four types of aggregate grains. Both presence and composition of grains are indicative of the different subfacies types. There is also a relation between grain composition and porosity of the Ca2-subfacies types. The size and quantity of ooids correlate positively with increasing porosity, whereas an increasing amount of algal structures (algal-lamination) correlates negatively with porosity. The Ca2-platform carbonates almost exclusively represent highstand systems tract and lowstand systems tract deposits. The presence or absence of type-3 aggregate grains within the grainy shoal and algal-laminated shoal subfacies allows the assignment of these subfacies to highstand (grains absent) or lowstand (grains present) systems tracts deposits. The Ca2-highstand deposits can be subdivided into four shallowing-upward parasequences (PS3 to PS7) bounded by parasequence boundaries (PSB3 to PSB6) and Zechstein sequence boundary ZSB4. In contrast to macroscopic core studies, microscopic studies to identify Ca2-subfacies types can utilize cutting material. This allows reconstruction of the subfacies distribution on the Ca2-platform, and delineation of potentially porous zones in uncored Ca2 intervals.     

Interactive effects of warming,eutrophication and size structure: impacts on biodiversity and food‐web structure

Warming and eutrophication are two of the most important global change stressors for natural ecosystems, but their interaction is poorly understood. We used a dynamic model of complex, size‐structured food webs to assess interactive effects on diversity and network structure. We found antagonistic impacts: Warming increases diversity in eutrophic systems and decreases it in oligotrophic systems. These effects interact with the community size structure: Communities of similarly sized species such as parasitoid–host systems are stabilized by warming and destabilized by eutrophication, whereas the diversity of size‐structured predator–prey networks decreases strongly with warming, but decreases only weakly with eutrophication. Nonrandom extinction risks for generalists and specialists lead to higher connectance in networks without size structure and lower connectance in size‐structured communities. Overall, our results unravel interactive impacts of warming and eutrophication and suggest that size structure may serve as an important proxy for predicting the community sensitivity to these global change stressors.     

Two dimensional diffusion of small molecules on protein surfaces: an EPR study of the restricted translational diffusion of protein-bound spin labels

Heisenberg spin exchange rates and dipole-dipole spin lattice relaxation rates for deuterated ¹⁴N- and ¹⁵N-spin labels bound selectively to the histidine His15 and to the lysines Lys13, 96, 97 of the lysozyme molecule have been determined with the aid of electron spin resonance spectroscopy. The results can be interpreted in terms of a two dimensional translational diffusion of the nitroxide tips of the spin labels along the protein surface within restricted surface areas. The spin labels are regarded as models for long amino acid side chains and as probes for the dynamics of protein and water in the vicinity of the protein surface. The translational diffusion coefficient DP_II is reduced by a factor of between six and thirty compared to the value of D found for the spin labels in bulk water, its value for T = 295 K is given by (1.3±0.6)·10^–10m²s^–1 D (2.4±0.3) 10^–11 m²s^–1. Offprint requests to: H.-J. Steinhoff     

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.     

Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.     

Spin labeling EPR

Photosynthesis Research - Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational...     

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function ( n = 11–14, P < 0.05) and decreased right ventricular wall stress ( n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 ± 1.1% versus 27.8 ± 1.2%; n = 6–8, P < 0.001) and greater capillary density (338.5 ± 14.7 versus 259.8 ± 9.2 vessels per mm; n = 6–8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 ± 0.03% versus 0.42 ± 0.03%; n = 6, P = 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit⁺ and CD34⁺ stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood ( n = 7, P < 0.05) and 48 hrs in the infarcted hearts ( n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts ( n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit⁺ and CD34⁺ stem cells via the enhanced expression of chemoattractant SDF-1.     

Microtubule Binding and Trapping at the Tip of Neurites Regulate Tau Motion in Living Neurons

During the development of neurons, the microtubule-associated tau proteins show a graded proximo-distal distribution in axons. In tauopathies such as Alzheimer's disease, tau accumulates in the somatodendritic compartment. To scrutinize the determinants of tau's distribution and motion, we constructed photoactivatable green fluorescent protein (GFP)-tagged tau fusion proteins and recorded their distribution after focal activation in living cells. Simulation showed that the motion of tau was compatible with diffusion/reaction as opposed to active transport/reaction. Effective diffusion constants of 0.7–0.8 μm²/second were calculated in neurites of PC12 cells and primary cortical neurons. Furthermore, tau's amino terminal projection domain mediated binding and enrichment of tau at distal neurites indicating that the tip of a neurite acts as an adsorber trapping tau protein. Treatment with taxol, incorporation of disease-related tau modifications, experimentally induced hyperphosphorylation and addition of preaggregated amyloid β peptides (Aβ) increased the effective diffusion constant compatible with a decreased binding to microtubules. Distal enrichment was present after taxol treatment but was suppressed at disease-relevant conditions. The data suggest that (i) dynamic binding of tau to microtubules and diffusion along microtubules and (ii) trapping at the tip of a neurite both contribute to its distribution during development and disease.     

The neurobiology of itch

The neurobiology of itch, which is formally known as pruritus, and its interaction with pain have been illustrated by the complexity of specific mediators, itch-related neuronal pathways and the central processing of itch. Scratch-induced pain can abolish itch, and analgesic opioids can generate itch, which indicates an antagonistic interaction. However, recent data suggest that there is a broad overlap between pain- and itch-related peripheral mediators and/or receptors, and there are astonishingly similar mechanisms of neuronal sensitization in the PNS and the CNS. The antagonistic interaction between pain and itch is already exploited in pruritus therapy, and current research concentrates on the identification of common targets for future analgesic and antipruritic therapy.