The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Brain proteomics identifies potential simvastatin targets in acute phase of stroke in a rat embolic model

Finding an efficient neuroprotectant is of urgent need in the field of stroke research. The goal of this study was to test the effect of acute simvastatin administration after stroke in a rat embolic model and to explore its mechanism of action through brain proteomics. To that end, male Wistar rats were subjected to a Middle Cerebral Arteria Occlusion and simvastatin (20 mg/kg s.c) (n = 11) or vehicle (n = 9) were administered 15 min after. To evaluate the neuroprotective mechanisms of simvastatin, brain homogenates after 48 h were analyzed by two‐dimensional fluorescence Difference in Gel Electrophoresis (DIGE) technology. We confirmed that simvastatin reduced the infarct volume and improved neurological impairment at 48 h after the stroke in this model. Considering our proteomics analysis, 66 spots, which revealed significant differences between groups, were analyzed by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry allowing the identification of 27 proteins. From these results, we suggest that simvastatin protective effect can be partly explained by the attenuation of the oxidative and stress response at blood–brain barrier level after cerebral ischemia. Interestingly, analyzing one of the proteins (HSP75) in plasma from stroke patients who had received simvastatin during the acute phase, we confirmed the results found in the pre‐clinical model.

     

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2^−/− mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.     

Breeding for flavour of fresh market tomato: sources for increasing acid content

Breeding programs in tomato for fresh consumption have concluded in very productive varieties/hybrids with an extraordinary external quality. However, internal quality has not been a priority objective in these breeding programs, so present products have a lack of important internal quality properties. Thus, internal and nutritional improvement including taste and organoleptyc characteristics are important breeding objectives at present. A screening trial to identify sources for internal quality improvement by increasing acid content was carried out. 12 accessions of Lycopersicon esculentum and 8 of L. pimpinellifolium were tested with that purpose. Content in citric, malic, oxalic and fumaric acid by HPLC, soluble solids content (SSC)(1 Brix) by refractometry and total acidity by titration with NaOH were measured. Sources for high citric, malic and fumaric acid content were found to begin those breeding programs. Results could possibly suggest an independent genetic control for every acid.     

Genetic variation and migration pathways of maritime pine (Pinus pinaster Ait) in the Iberian peninsula

The genetic variability and migration pathways of Pinus pinaster after glaciation in the Iberian peninsula was studied by means of 18 loci from 12 natural populations of the species. The analysis showed the existence of three groups of populations with different levels of diversity and patterns of recolonization. The southern Iberian group displays a high level of diversity, with a stepping-stone model of variation. The presence of rare alleles in this group and their position in the phylogenetic tree suggest the existence of refugia during glaciations in this zone. The eastern Iberian group also has high levels of diversity but is clearly separate from the first group based on their genetic distances. The Atlantic group displays a low level of diversity that could be interpreted as a rapid recolonization of the entire area by the Eastern group that has not yet developed to a divergence in this area. The southern Iberian range is indicated to be the dispersal centre of the species after the last glaciation. Received: 15 February 1999 / Accepted: 30 April 1999     

CO2 efflux from a Mediterranean semi-arid forest soil. II. Effects of soil fauna and surface stoniness

Many forest soils in the Mediterranean basin areshallow and contain high amounts of gravel in theorganic layers. Recent studies on soil organic matteraccumulation have shown high amounts of organic matteroccurring mainly in soils with high levels ofstoniness at the soil surface. The gravel layer mayaffect the microclimatic conditions of the soilsurface and probably the distribution and activity ofsoil fauna.In order to quantify the combined effects soil fauna(epigeic macrofauna and earthworms) and stoniness onthe release of soil CO₂, we performed a threefactor field experiment by using a series ofreconstructed soil profiles. Factors 1 and 2 consistedof the exclusion/presence of soil epigeic macrofaunaand earthworms, and factor 3 of the presence/absenceof a gravel layer intermingled with the H horizon. Weincubated ¹⁴C straw in the H horizon and carriedout three 40 mm rainfall simulations.Soil respiration primarily depended on the season. Theeffects of soil fauna were generally small and did notcoincide with periods of high faunal activity. Thelargest effects of both earthworms and soil epigeicfauna were found after wetting the soil in summer. Theeffects of the earthworms were concentrated in themineral soil while the effects of the epigeic faunawere concentrated in the H horizon and mainly arosetowards the end of the experiment. This suggests thatthe effects of epigeic fauna may have beenunderestimated due to the length of the experiment.The gravel layer increased the effect of faunaprobably by creating more favorable microclimaticconditions. The accumulation of organic matter insoils with high levels of stoniness cannot beexplained by the effect of gravel on soil microclimatenor by its effect on the activity of soil fauna.     

Competition for Milk in the Domestic Rabbit: Survivors Benefit from Littermate Deaths

We sought evidence for postnatal resource limitation among littermates of the domestic rabbit Oryctolagus cuniculus , and asked whether deaths of indi~vidual pups benefit survivors by increasing their share of milk. Milk ingestion, growth and mortality of 10 chinchilla-breed captive litters were recorded between birth and age 21 d. That milk limits growth and survival was indicated by larger litters showing lower weight gain and higher mortality, and by a significant positive correlation between milk ingested by individual pups and weight increase. Within litters, pups with higher birth weights grew faster, and weight hierarchies became increasingly stable over the 3 weeks, suggesting that advantages accrued during gestation were progressively consolidated during lactation. After individual pups died, the total daily milk weight obtained by the litter was generally unaffected but per capita milk consumption and growth of surviving pups increased, and increases in per capita consumption were greater in smaller litters. The most successful competitors for milk apparently benefit from the deaths of their littermates by obtaining an increased share of an undiminished daily food supply. This relationship has not previously been demonstrated in any vertebrate.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.