Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.     

Phosphoinositide 3-kinase signaling in the cellular response to oxidative stress

Oxidative stress is linked to the pathogenesis and pathobiochemistry of various diseases, including cancer, diabetes and cardiovascular disorders. The non-specific damaging effect of reactive oxygen species (ROS) generated during oxidative stress is involved in the development of diseases, as well as the activation of specific signaling cascades in cells exposed to the higher oxidant load. A cellular signaling cascade that is activated by several types of reactive oxygen species is the phosphoinositide 3'-kinase (PI 3-kinase)/protein kinase B (PKB) pathway, which regulates cellular survival and fuel metabolism, thus establishing a link between oxidative stress and signaling in neoplastic, metabolic or degenerative diseases. Several links of PI 3-kinase/PKB signaling to ROS are discussed in this review, with particular focus on the molecular mechanisms involved in the regulation of PI 3-kinase signaling by oxidative stress and important players such as (i) the glutathione and glutaredoxin system, (ii) the thioredoxin system and (iii) Ser/Thr- and Tyr phosphatases.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Modelling motor units in 3D: influence on muscle contraction and joint force via a proof of concept simulation

Functional heterogeneity is a skeletal muscle’s ability to generate diverse force vectors through localised motor unit (MU) recruitment. Existing 3D macroscopic continuum-mechanical finite element (FE) muscle models neglect MU anatomy and recruit muscle volume simultaneously, making them unsuitable for studying functional heterogeneity. Here, we develop a method to incorporate MU anatomy and information in 3D models. Virtual fibres in the muscle are grouped into MUs via a novel “virtual innervation” technique, which can control the units’ size, shape, position, and overlap. The discrete MU anatomy is then mapped to the FE mesh via statistical averaging, resulting in a volumetric MU distribution. Mesh dependency is investigated using a 2D idealised model and revealed that the amount of MU overlap is inversely proportional to mesh dependency. Simultaneous recruitment of a MU’s volume implies that action potentials (AP) propagate instantaneously. A 3D idealised model is used to verify this assumption, revealing that neglecting AP propagation results in a slightly less-steady force, advanced in time by approximately 20 ms, at the tendons. Lastly, the method is applied to a 3D, anatomically realistic model of the masticatory system to demonstrate the functional heterogeneity of masseter muscles in producing bite force. We found that the MU anatomy significantly affected bite force direction compared to bite force magnitude. MU position was much more efficacious in bringing about bite force changes than MU overlap. These results highlight the relevance of MU anatomy to muscle function and joint force, particularly for muscles with complex neuromuscular architecture.

     

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 μM. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

Solubilization and characterization of active neuropeptide Y receptors from rabbit kidney

Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.     

Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c₅₅₄; and cycB, cytochrome c_m552. The deduced protein sequences of HAO, c₅₅₄, and c_m552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c_m552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c₅₅₄ gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c₅₅₄ gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

Species richness of herbivores on exotic host plants increases with time since introduction of the host

Aim Species richness of insect herbivores feeding on exotic plants increases with abundance as well as range size of the host in the area of introduction. The formation of these herbivore assemblages requires a certain amount of time, and the richness of insect faunas should also increase with the length of time an exotic plant has been present in the introduced range. Location Central Europe. Methods We analysed the variation in species richness of leaf‐chewing Lepidoptera larvae and sap‐sucking Auchenorrhyncha (Hemiptera) associated with 103 exotic woody plant species in Germany in relation to time since introduction, range size, growth form (trees versus shrubs), biogeographical origin (distance from Central Europe) and taxonomic isolation of the host plant (presence or absence of a native congener in the introduced area). Results Using simple correlation analyses we found for Lepidoptera and Auchenorrhyncha that species richness increased with time since introduction of the host plant. For the Lepidoptera the increase of species richness with time since introduction remained significant even after removing the effects of all other independent variables. Main conclusions Our results provide some evidence that assemblages of insects on exotic plants do not reach saturation within a time scale of few hundred years. This contrasts with previous findings for crop plants.