We show here that human and mouse mesenchymal stem cells (MSCs) can be obtained not only from bone marrow (BM), but also from adult spleen and thymus. In vitro, both human and mouse spleen- and thymus-derived MSCs exhibit immunophenotypic characteristics and differentiation potential completely comparable to BM-MSCs. In addition, they can inhibit immune responses mediated by activated T lymphocytes with efficiency comparable to BM-MSCs. In vivo, mouse MSCs from BM, spleen, and thymus, if injected together with a genetically modified tumor cell vaccine, can equally prevent the onset of an anti-tumor memory immune response, thus leading to tumor growth in normally resistant mice. Our data suggest that not only do spleen and thymus have a stem cell reservoir to build up their stromal architecture, but also contain microenviromental immunoregulatory cells with the same properties of BM-MSCs.

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A new photosystem II preparation was isolated from Marchantia polymorpha thylakoids upon solubilization with dodecyl β-D-maltoside and glycerol gradient ultracentrifugation. Its protein composition was analyzed, and all tested polypeptides from the core complex, the oxygen-evolving enhancer and the lightharvesting complex (LHC) could be detected. The only component severely depleted compared with the grana membrane preparation was the psbS gene product. This complex was subjected to chemical crosslinking using the cleavable homobifunctional cross-linker dithiobis(sulfosuccinimidylpropionate). The overall pattern of cross-linking-products was analyzed by diagonal electrophoresis, where the cross-linking agent was cleaved by reduction of the disulfide bond between the first and second dimensions of the gel, followed by immunoblotting. Many cross-linking products were characterized and these data used in order to identify protein masses revealed by electron microscopy [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175Ϫ179]. lt is concluded that the core proteins CP43 and CP47 are located at opposite sides of the D1-D2-cytochrome b 559 complex. Minor CAB proteins were found to interact with core complex subunits (CP29, CP26) and LHCII (CP26), supporting the view that these proteins could interface the major LHCII with the reaction center. Photosystems of plants are located in the chloroplast mem-knowledge of distances between chromophores, mutual transition dipole orientation, absorption and fluorescence energy levbranes and are composed of a number of pigment-protein complexes each binding several chlorophyll molecules. The pho-els within individual chlorophyll-proteins. Moreover, knowledge of the topological organization of the pigment binding subunits tosystems are organized into two structurally and functionally distinct moieties : a core complex and an antenna. Core complex is required. In the case of photosystem (PS) II, progress has been made in understanding the structure of the reaction center on the chlorophyll-proteins are chloroplast encoded, bind chlorophyll a and β-carotene and catalyze the light-induced trans-membrane basis of its homology with the bacterial system (see Diner and Babcock, 1996, for a review). A model for the structure of the electron transport. The antenna system is composed of nuclearencoded polypeptides which are homologous to each other and major antenna complex (LHCII) at 3.4-Å resolution was obtained based on electron crystallography (Kühlbrandt et al., bind chlorophyll a, chlorophyll b and several xanthophyll spe-1994). Electron microscopy and electron crystallography proved cies. Light energy is mainly absorbed in the antenna and funextremely helpful in the analysis of the topological organization neled to the core complex. Nevertheless, antenna proteins also of the PSII supramolecular complex. Thus, the PSII core was have an extended regulative function thus acting in the control sh...

Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna lightharvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformationai changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stromaexposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transdnction pathway is discussed. 1, IntroductionIn the chloroplast, phosphorylation of membrane proteins i. responsible for many of the physiological responses to hanges in incident light and redox poise. The major substrate iavolved in this regulative process is the light-harvesting comllex of photosystem II (LHCII). When PSII is overexcited with respect to PSI, the plastoquinone pool becomes reduced i,nd thus activates a kinase bound to the cytochrome b6/f t omplex [l,2]. This process results in phosphorylation of the Jaajor trimeric LHCII complex in an N-terminal, stroma-exl,osed site [3] leading to the monomerisation of LHCII, dis-, onnection from PSII RC [2,4] and migration of LHCII ~aonomeric subpopulations from grana to stroma membranes t 2,5,6] where they can transfer excitation energy to PSI [5] and bus oxidise the plastoquinone pool. This process can there~ore be understood in terms of regulation of photosystem ntenna size by the transfer of LHCII units away from the Corresponding author. Fax: (39) 45-8098929. ~-mail: bassi@biotech.univr.it tbbreviations. BBY, PSII membrane preparation; CK2, casein kinase !; Chl, chlorophyll; CP, chlorophyll-protein; DM, dodecyl-maltoside; EDTA, ethylenediaminetetraacetic acid; ELFE, electroendoosmotic ~'lectrophoresis; HEPES, N-2-(hydroxy-ethyl)piperazine-N'-2-ethane~ulfonic acid; IEF, isoelectrofocusing; LHCII, light-harvesting complex of PSII; PAGE, polyacrylamide gel electrophoresis; PS, photosystem; rCP29, recombinant CP29 reconstituted from the apoprotein derivatives overproduced in bacteria; RC, reaction centre; ~;DS, sodium dodecyl sulphate; Tris, 2-amino-2-(hydroxymethyl)-l,3-l~ropanediol overexcited photosystem [2]. The underlying mechanism is a conformational change of the LHCII N-terminal portion which undocks this subunit from PSII and increases its affinity for PSI [2].More recently, it was reported that photoinhibitory conditions induce reversible phosphorylation of CP29, another PSII subunit PSI [7]. Although there is strong homology between CP29 and LHCII and both protei...

Summary De novo expression of costimulatory molecules in tumours generally increases their immunogenicity, but does not always induce a protective response against the parental tumour. This issue was addressed in the mouse Sp6 hybridoma model, comparing different immunization routes (subcutaneous, intraperitoneal and intravenous) and doses (0·5 × 106 and 5 × 106 cells) of Sp6 cells expressing de novo B7‐1 (Sp6/B7). The results can be summarized as follows. First, de novo expression of B7‐1 rendered Sp6 immunogenic, as it significantly reduced the tumour incidence to ≤15% with all delivery routes and doses tested, whereas wild‐type Sp6 was invariably tumorigenic (100% tumour incidence). Second, long‐lasting protection against wild‐type Sp6 was mainly achieved when immunization with Sp6/B7 was subcutaneous: a dose of 0·5 × 106 Sp6/B7 cells elicited protection that was confined to sites in the same anatomical quarter as the immunizing injection. Repeated injections of the same dose extended protection against wild‐type Sp6 to other anatomical districts, as well as a single injection of a 10‐fold higher dose (5 × 106 cells). Finally, Sp6‐specific cytotoxic T‐lymphocyte activity was detected in draining lymph nodes, and the splenic expansion of Sp6‐specific cytotoxic T‐lymphocyte precursors quantitatively correlated with the dose of antigen. We conclude that activation of a protective immune response against Sp6 depends on the local environment where the immunogenic form of the ‘whole tumour cell antigen’ is delivered. The antigen dose regulates the anatomical extent of the protective response.

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The hypothesis that phosphorylation of the minor photosystem II antenna complex CP29 (CP34 formation) in Zea mays (cv. Dekalb DK300), under conditions of illumination and low temperature stress, may constitute a protective mechanism against photoinhibition, has been investigated. It is demonstrated that illumination at low temperature induces a marked increase in reversible non‐photochemical quenching yield of chlorophyll fluorescence, together with CP34 formation. These two parameters, however, are not related as CP34 dephosphorylates to CP29 in the dark, with a half‐time of about 10 min, while the enhanced non‐photochemical quenching yield is stable for many hours. The enhanced non‐photochemical quenching yield seems to correlate with zeaxanthin formation. The influence of CP34 formation on photoinhibition was also directly investigated. No measurable effect on this parameter could be observed after treatment with high light. It is concluded that CP34 is probably not directly involved in photoprotective processes.