An alternative carotenoid-to-bacteriochlorophyll energy transfer pathway in photosynthetic light harvesting

Blue and green sunlight become available for photosynthetic energy conversion through the light-harvesting (LH) function of carotenoids, which involves transfer of carotenoid singlet excited states to nearby (bacterio)chlorophylls (BChls). The excited-state manifold of carotenoids usually is described in terms of two singlet states, S(1) and S(2), of which only the latter can be populated from the ground state by the absorption of one photon. Both states are capable of energy transfer to (B)Chl. We recently showed that in the LH1 complex of the purple bacterium Rhodospirillum rubrum, which is rather inefficient in carotenoid-to-BChl energy transfer, a third additional carotenoid excited singlet state is formed. This state, which we termed S*, was found to be a precursor on an ultrafast fission reaction pathway to carotenoid triplet state formation. Here we present evidence that S* is formed with significant yield in the LH2 complex of Rhodobacter sphaeroides, which has a highly efficient carotenoid LH function. We demonstrate that S* is actively involved in the energy transfer process to BChl and thus have uncovered an alternative pathway of carotenoid-to-BChl energy transfer. In competition with energy transfer to BChl, fission occurs from S*, leading to ultrafast formation of carotenoid triplets. Analysis in terms of a kinetic model indicates that energy transfer through S* accounts for 10-15% of the total energy transfer to BChl, and that inclusion of this pathway is necessary to obtain a highly efficient LH function of carotenoids.     

Initial electron donor and acceptor in isolated Photosystem II reaction centers identified with femtosecond mid-IR spectroscopy

Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm(-1), with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6- to 0.8-ps time scale, whereas P+, the precursor state for water oxidation, is formed after approximately 6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the "accessory chlorophyll" Chl(D1) and the pheophytin on the so-called active branch.     

The long-range organization of a native photosynthetic membrane

Photosynthesis relies on the delicate interplay between a specific set of membrane-bound pigment-protein complexes that harvest and transport solar energy, execute charge separation, and conserve the energy. We have investigated the organization of the light-harvesting (LH) and reaction-center (RC) complexes in native bacterial photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides by using polarized light spectroscopy, linear dichroism (LD) on oriented membranes. These LD measurements show that in native membranes, which contain LH2 as the major energy absorber, the RC-LH1-PufX complexes are highly organized in a way similar to that which we found previously for a mutant lacking LH2. The relative contribution of LH1 and LH2 light-harvesting complexes to the LD spectrum shows that LH2 preferentially resides in highly curved parts of the membrane. Combining the spectroscopic data with our recent atomic force microscopy (AFM) results, we propose an organization for this photosynthetic membrane that features domains containing linear arrays of RC-LH1-PufX complexes interspersed with LH2 complexes and some LH2 found in separate domains. The study described here allows the simultaneous assessment of both global and local structural information on the organization of intact, untreated membranes.     

SPI-CI and SPI-6 cooperate in the protection from effector cell-mediated cytotoxicity

Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrM-induced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cells.     

The prognostic significance of early treatment response in pediatric relapsed acute myeloid leukemia: results of the international study Relapsed AML 2001/01

The prognostic significance of early response to treatment has not been reported in relapsed pediatric acute myeloid leukemia. In order to identify an early and easily applicable prognostic factor allowing subsequent treatment modifications, we assessed leukemic blast counts in the bone marrow by morphology on days 15 and 28 after first reinduction in 338 patients of the international Relapsed-AML2001/01 trial. Both day 15 and day 28 status was classified as good (≤20% leukemic blasts) in 77% of patients. The correlation between day 15 and 28 blast percentages was significant, but not strong (Spearman correlation coefficient = 0.49, P<0.001). Survival probability decreased in a stepwise fashion along with rising blast counts at day 28. Patients with bone marrow blast counts at this time-point of ≤5%, 6–10%, 11–20% and >20% had 4-year probabilities of survival of 52%±3% versus 36%±10% versus 21%±9% versus 14%±4%, respectively, P<0.0001; this trend was not seen for day 15 results. Multivariate analysis showed that early treatment response at day 28 had the strongest prognostic significance, superseding even time to relapse (< or ≥12 months). In conclusion, an early response to treatment, measured on day 28, is a strong and independent prognostic factor potentially useful for treatment stratification in pediatric relapsed acute myeloid leukemia. This study was registered with ISRCTN code: 94206677.     

Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.     

Tumor Eradication by Wild-type p53-specific Cytotoxic T Lymphocytes

The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies. The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility complex class I molecule H-2K^b, were generated by immunizing p53 gene deficient (p53 −/−) C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue. When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for weeks in the absence of immunopathology and were capable of preventing tumor outgrowth. Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can serve as a target for CTL-mediated immunotherapy of tumors.The efficacy of virus-specific CTLs to eradicate virus-induced tumors has been well documented (1–5). However, since the majority of tumors are not virus- induced, characterization of tumor-associated antigens encoded by cellular genes is important for the development of new immunotherapeutic strategies. Target antigens on nonvirally-induced tumors recognized by CTLs were recently identified, notably in patients with melanoma (6–18). The fact that these antigens are lineage- or tumor-specific, limits the use of these targets in immunotherapy to a small group of cancers. On the other hand, the expression of some of these antigens on normal melanocytes demonstrates that self antigens can serve as targets for CTL-mediated destruction of tumors.Mutations in the gene encoding the tumor suppressor protein p53 are found in ∼50% of all human malignancies (19). Recently, a direct link has been established between mutational hot spots in the p53 gene leading to its overexpression, and carcinogenic metabolites derived from agents in cigarette smoke (20). In normal cells, p53 induces a cell cycle arrest, allowing DNA to be checked for irregularities, thereby guarding the integrity of the genome (21). Mutation of p53 abolishes its function as a suppressor of the cell cycle, promoting the escape of transformed cells from the normal restriction of controlled growth. Since these mutations, causing overexpression of p53, are present in a wide variety of cancers (22–25), a large group of patients would benefit from p53 directed immunotherapy. One could consider mutant p53 sequences as target antigens for tumor-specific CTLs. However, p53 mutations occur at many different sites in the p53 molecule, necessitating identification of the site of mutation in each patient before therapy. Furthermore, not all mutations are contained in MHC-binding CTL epitopes. If, in contrast, wild-type (wt)¹ p53 sequences are used, the entire sequence of the p53 protein is available for properly processed immunogenic T cell epitopes. We hypothesized that the altered expression of p53, seen in many cancers, leads to modified processing and presentation of wt p53-derived peptides by MHC class I molecules. Recently, wt p53 peptide-specific CTL were generated from human and murine responding lymphocytes, some of which recognized p53-overexpressing tumors in vitro (26–34).Here, we report the in vivo eradication of established p53-overexpressing tumors in C57BL/6 p53 (+/+) nude mice by a well-defined wt p53-specific CTL clone in the absence of any demonstrable immunopathology. These CTLs, generated in p53-deficient mice and recognizing the murine wt p53-derived epitope AIYKKSQHM (amino acids [aa] 158–166) presented by the MHC class I molecule H-2K^b, were also capable of preventing the outgrowth of a more aggressive p53-overexpressing tumor in immunocompetent p53 (+/+) C57BL/6 mice.     

Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism

OBJECTIVE: To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation. METHODS: Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n = 10) and patients with other forms of arthritis (non-RA; n = 10) and in plasma from healthy subjects (n = 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F(1+2) (by enzyme-linked immunosorbent assay [ELISA]) and thrombin-antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo. RESULTS: Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non-RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F(1+2) (median 66 nM) and TAT complex (median 710 microg/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 microg/liter, respectively) as well as the controls (1.0 nM and 2.9 microg/liter, respectively). CONCLUSION: Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII-dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.     

Diagnosis, treatment and prevention of ankle sprains: an evidence-based clinical guideline

Ankle injuries are a huge medical and socioeconomic problem. Many people have a traumatic injury of the ankle, most of which are a result of sports. Total costs of treatment and work absenteeism due to ankle injuries are high. The prevention of recurrences can result in large savings on medical costs. A multidisciplinary clinical practice guideline was developed with the aim to prevent further health impairment of patients with acute lateral ankle ligament injuries by giving recommendations with respect to improved diagnostic and therapeutic opportunities. The recommendations are based on evidence from published scientific research, which was extensively discussed by the guideline committee. This clinical guideline is helpful for healthcare providers who are involved in the management of patients with ankle injuries.