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 chromophorebinding 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.Light energy for photosynthesis of green plants is collected by an antenna system composed of many homologous proteins belonging to the Lhc (light-harvesting complex) multigene family (1). These pigment-protein complexes are organized around photosynthetic reaction centers to form supramolecular complexes embedded in the thylakoid membranes and account for Ϸ70% of the pigments involved in photosynthesis. Understanding of energy-transfer processes in the antenna and reaction centers requires knowledge of the topological organization of subunits (2-4), of the distances between chromophores, and of their mutual transition-dipole orientation and the absorption/ fluorescence energy levels. Although the resolution of the photosystem II LHC structure (LHCII) at 3.4-Å resolution (5) has allowed localization of chlorophyll (Chl)-binding sites and their relative distances, identification of transition-dipole orientation and energy levels are precluded by insufficient resolution of the structure thus far obtained or are not accessible by using structural analysis. We have used an alternative approach for the identification of the Chl-a, Chl-b, and xanthophyll molecules among the different binding sites and for determination of their individual absorption spectra: a series of mutant apoproteins was constructed by overexpression in bacteria of the Lhcb4 gene, in which individual chlorophyll-binding residues (5) were substituted for by residues unable to coordinate porphyrins. On in vitro refolding with purified pigments, proteins missing individual chromophores were obtained for seven of eight chlorophyllbinding sites and for one of the two xanthophyll-binding sites present in this antenna protein. B...

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day −7) or double (portal vein, day −7 and tail vein, day −1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4+CD25+Foxp3+ Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.

؉ CD25 high cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus-but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4 ؉ CD25 high Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.

The minor light-harvesting chlorophyll-alb-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, 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 moleculeslpolypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-alb-binding protein of photosystem I1 (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, Keywords: photosynthesis ; Lhcb4; xanthophyll proteins.In the chloroplasts of higher plants, chlorophyll and carotenoid molecules are non-covalently bound to specific transmembrane proteins. These represent antenna complexes and are called light-harvesting chlorophyll-ah-binding proteins of photosystem I and I1 (LHCI and LHCII, respectively). Light is harvested by these two antenna complexes and excitation energy is delivered to the reaction centres of photosystem I (PSI) and photosystem I1 (PSII), where transmembrane electron transport occurs, generating a trans-thylakoid pH gradient, ATP synthesis and NADP' reduction.Photosystem I1 light harvesting complex (LHC) is composed of four chlorophyll-alb-binding proteins : the major LHCII, binding approximately 65 % of PSII chlorophyll, and the three minor complexes called CP24, CP26 and CP29 which, all together, bind about 15% of total PSII chlorophyll Peter and Thornber, 1991 ; Jansson et al., 1992). Due to the low amount of bound pigments, it seems unlikely that the main function of the minor complexes is to harvest light. On the contrary, several lines of evidence indicate that they are involved in the regulation of the level of chlorophyll a excited states. Such regulation is required to prevent overexcitation and photoinhibition of PS 11. At least 80% of the xanthophyll violaxanthin is located in minor complexes in maize Adams, 1992). Non-photochemical quenching operates through a xanthophyll cycle, that includes the deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Bassi and Yamamoto, 1995). The involvement of minor complexes in regulation mechanisms is also supported by the binding of the NPQ inhibitor dicychlohexylcarbodiimide [(cHxN),C] to CP26 and CP29 and by the finding that photoinhibitory conditions lead to a conformational change of CP29 caused by phosphorylation. The phosphorylation takes place in cold-resistant, but not in cold-sensitive, maize plants . Finally, the location of the minor complexes between the reaction centre and the major LHCII complex is well placed for regulating excitation-energy supply to, or diversion from, PSII.The major anten...

Background: Microalbuminuria is an early sign of kidney disease in diabetes and indicates cardiovascular risk. We tested if a prespecified urinary proteomic risk classifier (CKD273) was associated with development of microalbuminuria and if progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone. Methods: Prospective multicentre study in people with type 2 diabetes, normal urinary albumin excretion and preserved renal function in 15 European specialist centres. High-risk individuals determined by CKD273 were randomised 1:1 (interactive web response system) in a double-blind randomised controlled trial comparing spironolactone 25 mg o.d. to placebo. Primary endpoint was development of confirmed microalbuminuria in all individuals with available data. Secondary endpoints included reduction in incidence of microalbuminuria with spironolactone and association between CKD273 and impaired renal function defined as a glomerular filtration rate < 60 ml/min per 1•73 m 2. This study is registered with ClinicalTrials.gov: NCT02040441 and is completed. Findings: From March 25, 2014 to September 30, 2018 we followed 1775 participants, 12% (n=216) had high-risk urinary proteomic pattern of which 209 were included in the trial and assigned spironolactone (n=102) or placebo (n=107). Median follow-up time was 2•51 years (IQR 2•0-3•0). Progression to microalbuminuria was seen in 28•2% of high-risk and 8•9% of low-risk people (P< 0•001) (hazard ratio (HR), 2•48; 95% confidence interval [CI], 1•80 to 3•42 P<0•001, independent of baseline clinical characteristics). A 30% decline in eGFR from baseline was seen in 42 (19•4 %) high-risk participants compared to 62 (3•9 %) low-risk participants, HR 5•15; 95 % CI (3•41 to 7•76; p<0.0001). Development of microalbuminuria was seen in 35 (33%) randomised to placebo and 26 (25%) randomised to spironolactone treatment (HR 0•81, 95% CI, 0•49 to 1•34, P=0•41). Harms: hyperkalaemia was seen in 13 versus 4, and gynaecomastia in 3 versus 0 subjects on spironolactone and placebo, respectively. Interpretation: In people with type 2 diabetes and normoalbuminuria, the urinary proteomic classifier CKD273 was associated with a 2•5 times increased risk for progression to microalbuminuria over a median of 2•5 years, independent of clinical characteristics. Spironolactone did not prevent progression to microalbuminuria in high-risk subjects.

The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

Repertaxin 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.

These data suggest that, despite enhanced Treg expression, low-dose SRL combined to alemtuzumab induction and MMF-based steroid-free maintenance therapy, does not appreciably protect renal transplant recipients from chronic allograft injury and dysfunction.