Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/reperfusion

Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/reperfusion.BackgroundIschemia/reperfusion (I/R) injury after organ transplantation is a major cause of delayed graft function. Following I/R, locally produced CXC chemokines attract and activate granulocytes, which in turn promote graft damage.MethodsWe examined the involvement of granulocyte recruitment via the CXCR2 pathway in a rat model of 4 hours cold ischemia followed by kidney transplantation. Serum creatinine and intragraft granulocyte infiltration were monitored in the early phase posttransplant. A CXCR2 inhibitor, repertaxin, was given to recipients before transplantation (at -24 hours or -8 hours or -2 hours), immediately before reperfusion and 2 hours later.ResultsAn increase of granulocyte chemoattractant CINC-1/interleukin-8 (IL-8) mRNA expression after I/R both in syngeneic and allogeneic transplantation was associated with a marked infiltration of granulocytes in renal tissue. In syngeneic transplantation, Lewis rats given 15 mg/kg repertaxin 24 hours before surgery had granulocyte graft infiltration and serum creatinine levels significantly reduced in respect to vehicle-treated animals. Intermediate effects were observed with 5 mg/kg, whereas the dose of 30 mg/kg had toxic effects. We found that reducing the pretreatment time to 8 hours before surgery was still effective. Prevention of granulocyte infiltration and serum creatinine increase was also obtained in allogeneic transplantation, when Brown Norway recipients of Lewis kidneys were given 15 mg/kg repertaxin starting 8 hours before surgery.ConclusionRepertaxin treatment of the recipient animal was effective in preventing granulocyte infiltration and renal function impairment both in syngeneic and in allogeneic settings. The possibility to modulate I/R injury in this rat model opens new perspectives for preventing posttransplant delayed graft function in humans

Reconstitution and pigment-binding properties of recombinant CP29

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coil, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for: the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native One, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, E G. and Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-1501 Paulsen, H., Rumler, U. and Rudiger, W (1990) Planta (Neidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII

Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a–Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins

Human in the Loop: A Model to Integrate Interaction Issues in Complex Simulations

Several activities of the product development process as for example ergonomic analyses, usability testing, and what is defined as User Experience - UX- design in general require humans to be involved as testers. In order to achieve a good effectiveness degree, these tests must be performed on prototypes as much as possible similar to the final product, and this is costly and sometimes difficult to obtain during the development process. This is especially true at the earliest stages of the process. Functional mock-up - FMU - methods and tools can be of great help, because they allow technological aspects of the products, as electronics, hydraulics, mechanics, etc. to be represented and managed in a simple and effective way. Mathematical equations allow product behavior to be determined, due to input values representing the application environment of the product. At the moment, an FMU model is great in simulating product behavior from the technological point of view, but concerns about user interaction issues are left apart. The research described in this paper aims at widening the coverage of FMU to user-product interaction issues. The goal aims at evaluating the possibility of substituting real users with a characterization of them, and to model and simulate interaction in a homogeneous way together with all the other product aspects. All of this makes the research activities very challenging, and the result is a sort of FMU-assisted interaction modeling. As an evolution of what is generally recognized as hardware and software-in-the-loop, this methodology will be referred as human-in-the-loop