Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.
Moderate heat stress (40°C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40°C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved. Photosystem II (PS II)3 in higher plants is a multisubunit complex composed of more than 25 proteins and the associated cofactors. Excitation energy captured by the chlorophylls and carotenoids in the light-harvesting chlorophyll protein complexes of PS II is finally transferred to P680, the reaction center chlorophyll of PS II, where charge separation takes place. In particular, PS II performs oxidation of water and reduction of plastoquinone molecules via chlorophyll-mediated photochemical reactions. Although it plays such an important role in the primary photochemical reaction of photosynthesis, PS II is vulnerable to various environmental stresses such as excessive visible light and high temperature.When irradiated with excessive visible light, the D1 protein is oxidatively damaged, and electron transport is inhibited. This process is referred to as photoinhibition of PS II (1-4). The photo-damaged D1 protein is subsequently degraded by specific proteases (5), and the repair of PS II is accomplished by the integration of a newly synthesized D1 protein to the PS II complex (6). Photoinhibition of PS II is caused by either the socalled acceptor-side or donor-side mechanism or both (4, 7). The acceptor-side photoinhibition takes place when the acceptor side of PS II is over-reduced by excessive illumination and the double-reduced Q A molecule is released from its binding site. Reversed electron flow from the primary electron acceptor pheophytin to P680 in the absence of Q A generates the triplet state P680, which reacts with molecular oxygen to form singlet oxygen ( 1 O 2 ). The 1 O 2 eventually damages the nearby polypeptide, the D1 protein. Alternatively, oxygen molecules may be reduced at the acceptor side of PS II to produce superoxide anion radicals (O 2 . ), which are turned into hydrogen peroxide (H 2 O 2 ) and finally hydroxyl radical (HO ⅐ ) through the Fenton reaction (8). It is claimed, however, that the generation of...
Background: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibodymediated thrombocytopenia. Study Design and Methods: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to highresolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. Results: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. Conclusion: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.
Background Alloantibodies against human platelet antigen (HPA)‐15 are sometimes detected in patients with platelet transfusion refractoriness (PTR); however, little is known about their impact on PTR. Study Design and Methods Two patients who possessed HPA‐15 alloantibodies (Patient 1, anti‐HPA‐15b; Patient 2, anti‐HPA‐15a) and human leukocyte antigen (HLA) antibodies were enrolled. The efficacy of HPA‐15–compatible vs –incompatible platelet transfusion was compared by focusing on ABO‐ and HLA‐matched transfusions on the basis of the 24‐hour corrected count increment (CCI‐24 hours) for platelets. The titers of HPA‐15 antibodies in the patients' sera were also monitored. Results The patients received 71 and 12 ABO‐compatible, HLA‐matched platelet transfusions, respectively, during the monitoring periods. Among these transfusions, CCI‐24 hours could be calculated in 27 and 10 transfusions, respectively, and the HPA‐15 genotype of the donors was determined. There were no significant differences in the CCI‐24 hours between the HPA‐15 compatible and incompatible transfusions in both patients (P = .30 and .56, respectively, Mann‐Whitney U test). There was no significant change in the HPA‐15b antibody titer in Patient 1 during the monitoring period, while the HPA‐15a antibody level in Patient 2 was undetectable at the end of the monitoring period, although the titer was low at the beginning. Conclusion The efficacy of HPA‐15–incompatible platelet transfusions was not necessarily inferior to that of HPA‐15 compatible ones. Although the case number was limited, our results suggest that HPA‐15 antibodies do not have a significant impact on the effects of platelet transfusion.
Background and objectives Although HLA‐eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process’ efficiency by employing a hollow‐fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation. Materials and methods HLA‐eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow‐fibre system. The efficiency of this platelet recovery process was determined. The resulting products’ platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals. Results The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody‐containing sera, suggested that HLA‐ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist‐induced aggregability and adherence/aggregability of the collagen‐coated plates under certain conditions, were conserved and not significantly different from the original ones. Conclusion We propose a novel preparation system for producing HLA‐eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality.
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