Astrocytes and stroke: networking for survival?

Astrocytes are now known to be involved in the most integrated functions of the central nervous system. These functions are not only necessary for the normally working brain but are also critically involved in many pathological conditions, including stroke. Astrocytes may contribute to damage by propagating spreading depression or by sending proapoptotic signals to otherwise healthy tissue via gap junction channels. Astrocytes may also inhibit regeneration by participating in formation of the glial scar. On the other hand, astrocytes are important in neuronal antioxidant defense and secrete growth factors, which probably provide neuroprotection in the acute phase, as well as promoting neurogenesis and regeneration in the chronic phase after injury. A detailed understanding of the astrocytic response, as well as the timing and location of the changes, is necessary to develop effective treatment strategies for stroke patients.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency--involvement of protein kinase A type I

Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4(+)/CD8(+), CD45RA(+)/RO(+), or CD28(-) T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.     

Superantigen-induced regulatory T cells display different suppressive functions in the presence or absence of natural CD4+CD25+ regulatory T cells in vivo

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4(+)CD25(+) regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4(+)CD25(+) regulatory T cells. In this study, we demonstrate that CD4(+)CD25(+) as well as CD4(+)CD25(-) T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4(+)CD25(+) regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4(+) T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4(+)CD25(-) T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.     

Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.     

Genetic variation in three species of Hordeum,and the selection of accessions for the Barley Core Collection

Starch gel electrophoresis of isozymes was used to investigate the level and distribution of genetic variation in accessions of three wild, diploid Hordeum species. Allelic variation was assessed for seven isozyme loci and used in the statistical analysis of 18 accessions of Hordeum brachyantherum subsp. californicum, 29 accessions of H. murinum subsp. glaucum and 11 accessions of H. pusillum. A major purpose with this study was to evaluate the selection of accessions for the international Barley Core Collection, complementing existing passport data. Allelic variation was found in all three species. The variation was mainly between accessions, as most accessions were fixed for certain alleles. Since all three species are self-pollinated, this was anticipated. The fact that the accessions have undergone one or several regeneration cycles from a rather limited number of individuals, after being collected, likely also have reduced the variation. The accessions were clustered by subjecting the genetic distances between them to UPGMA statistical treatment. The clusters in the dendrograms displayed for H. brachyantherum subsp. californicum and H. pusillum distinct patterns related to the geographical origin of the accessions. The accessions of H. murinum subsp. glaucum were less variable than the other two, and did not reveal such a clear pattern. By comparing the already made, geographically based, selection of accessions with the results from this study, it was evident that the selection had worked fairly well for H. brachyantherum subsp. californicum, but less so for the other two species.     

A single nucleotide polymorphism in the promoter region of the human gene for osteoprotegerin is related to vascular morphology and function

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and has previously been shown to regulate bone mass by inhibiting osteoclast differentiation and activation. Recent evidence indicates that OPG also plays a role in the vascular system, since ablation of the OPG gene in mice results in calcification of the aorta and renal arteries, and association has been found between serum levels of OPG and cardiovascular mortality. This study presents a novel single nucleotide polymorphism, a T/C transition located 129 bp upstream the TATA-box of the human OPG gene, detected by sequence analysis. The OPG genotype was determined by restriction fragment length polymorphism in a cohort consisting of 59 healthy subjects. The intima-media thickness (IMT) in the common carotid artery and maximal post-ischemic forearm blood flow (FBF) were investigated. Subjects with the CC genotype showed a significantly increased IMT (p<0.05) and a concommitantly reduced maximal FBF (p<0.01) as compared to those with the T allele. Thus, our results show that the polymorphism in the promoter region of OPG is associated with both vascular morphology and function in apparently healthy subjects.     

Potential applications and pitfalls of Bayesian inference of phylogeny

Only recently has Bayesian inference of phylogeny been proposed. The method is now a practical alternative to the other methods; indeed, the method appears to possess advantages over the other methods in terms of ability to use complex models of evolution, ease of interpretation of the results, and computational efficiency. However, the method should be used cautiously. The results of a Bayesian analysis should be examined with respect to the sensitivity of the results to the priors used and the reliability of the Markov chain Monte Carlo approximation of the probabilities of trees.