Proteomic analysis of mouse brain cortex identifies metabolic down-regulation as a general feature of ischemic pre-conditioning

J. Neurochem. (2012) 122, 1219-1229. ABSTRACT: The molecular mechanisms that lead to ischemic pre-conditioning are not completely understood, and proteins are important players. We compared the mouse brain cortex proteome from different ischemia sets: transient (7?min) middle cerebral artery occlusion (7'MCAo, pre-conditioning stimulus), permanent MCAo (pMCAo, severe ischemia), and pMCAo 4?days after 7'MCAo (7'MCAo/pMCAo, pre-conditioned model). Proteins were analyzed by two-dimensional electrophoresis coupled to liquid chromatography-tandem mass spectrometry. Overall, 28 proteins were expressed differentially from sham controls, and identified. The ischemic pre-conditioning stimulus alone up-regulated the stress protein heat-shock protein 70 (HSP70), possibly activated by the androgen receptor. Western blotting confirmed the increased expression of HSP70 and showed that androgen receptor expression paralleled that of HSP70. In the ischemic-tolerant group (7'MCAo/pMCAo), a number of proteins over-expressed after pMCAo returned to sham levels, seven proteins remained up-regulated as in pMCAo, and five proteins mainly involved in energy metabolism and mitochondrial electron transport and unchanged in pMCAo were down-regulated only in ischemic tolerance, suggesting a role in brain pre-conditioning. Astrocytes participated in ischemic-tolerance induction, as shown by the down-regulation of glutamine synthetase in the 7'MCAo/pMCAo group. The results suggest that metabolic down-regulation was a general feature of ischemic pre-conditioning, playing a pivotal role in neuroprotection.     

Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.     

Role of the calpain-calpastatin system in the density-dependent growth arrest

In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol.In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of α- and θ-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.     

D8S7 is consistently deleted in inverted duplications of the short arm of chromosome 8 (inv dup 8p)

Ten patients with inverted duplication of 8p (inv dup 8p) were studied with cytogenetic, biochemical and molecular techniques. The duplication for the region 8p12-p22 was always associated with a deletion of the locus D8S7 (mapped in 8p23.1) as demonstrated with the probe pSW50 by both in situ hybridization and Southern blot. Restriction fragment length polymorphisms detected by probes pSW50 (1 case) and by pG2LPL35 (locus LPL) (two cases) were informative as to a maternal origin of the anomaly. The activity of glutathione reductase, whose gene maps in the duplicated region at 8p21.1, was increased in all patients. The recognizable phenotype of inv dup 8p includes neonatal hypotonia, prominent forehead, large mouth with everted lower lip, abnormally shaped large ears, brain malformations and severe mental retardation. Our findings indicate that the chromosome rearrangement is homogeneous at least for the presence of the deletion and support the hypothesis of a common mechanism of origin.     

Tumor-peptide-pulsed dendritic cells isolated from spleen or cultured in vitro from bone marrow precursors can provide protection against tumor challenge

 Dendritic cells (DC) purified from murine spleen or generated in vitro from bone marrow precursors were compared for their respective abilities to stimulate T cell responses and provide tumor protection in vivo. In vitro incubation with synthetic tumor peptide conferred on both DC populations the ability to induce proliferation of tumor-peptide-specific T cells in vitro. Spleen DC were reproducibly about twofold more effective than bone-marrow-derived DC in this assay. Both DC populations could also induce cytotoxic activity in vivo. In vitro cytoxicity assays showed that, while cytotoxic activity induced by immunization with spleen DC was clearly peptide-specific, a high non-specific cytotoxic activity was consistently observed after immunization with bone-marrow-derived DC, whether peptide-pulsed or not. Regardless of such high non-specific activity in vitro, only tumor-peptide-pulsed DC could provide protection against subsequent inoculation of tumor cells. DC not pulsed with tumor peptide were ineffective. We conclude that DC isolated from spleen or generated in vitro from bone marrow precursors are suitable reagents for use in tumor vaccination studies. Received: 13 March 1997 / Accepted: 25 May 1997     

Conversion of allyl alcohol into acrolein by rat liver

1. Acrolein was detected in rat liver suspensions incubated in the presence of allyl alcohol and allyl formate. Acrolein was determined by condensation of the distilled aldehyde with semicarbazide, followed by spectrophotometric measurement of the semicarbazone at 257nm and identification by paper chromatography. 2. The transformation of allyl alcohol into acrolein occurred in the presence of NAD(+). Inhibitors of rat liver alcohol dehydrogenase inhibited the reaction. 3. It is suggested that the hepatotoxic actions of allyl alcohol and of allyl formate on rat liver are related to their conversion into acrolein.     

Urease production and DNA-homology in the species Bifidobacterium suis

Summary Urease production in the species Bifidobacterium suis was studied. The strains examined were strictly homogeneous in their DNA homology relationships. Most strains (74%) possess this enzyme. The presence of urease is therefore of value as additional diagnostic character of this species.