In Vitro Synthesis of Chlorophyll A in the Dark Triggers Accumulation of Chlorophyll A Apoproteins in Barley Etioplasts”

An in vitro translation system using lysed etioplasts was developed to test if the accumulation of plastid-encoded chlorophyll a apoproteins is dependent on the de novo synthesis of chlorophyll a. The P700 apoproteins, CP47 and CP43, were not radiolabeled in pulsechase translation assays employing lysed etioplasts in the absence of added chlorophyll precursors. When chlorophyllide a plus phytylpyrophosphate were added to lysed etioplast translation assays in the dark, chlorophyll a was synthesized and radiolabeled P700 apoproteins, CP47 and CP43, and a protein which comigrates with D1 accumulated. Chlorophyllide a or phytylpyrophosphate added separately to the translation assay in darkness did not induce chlorophyll a formation or chlorophyll a apoprotein accumulation. Chlorophyll a formation and chlorophyll a apoprotein accumulation were also induced in the lysed etioplast translation system by the photoreduction of protochlorophyllide to chlorophyllide a in the presence of exogenous phytylpyrophosphate. Accumulation of radiolabeled CP47 was detectable when very low levels of chlorophyll a were synthesized de novo (less than 0.01 nmol/10(7) plastids), and radiolabel increased linearly with increasing de novo chlorophyll a formation. Higher levels of de novo synthesized chlorophyll a were required prior to detection of radiolabel incorporation into the P700 apoproteins and CP43 (greater than 0.01 nmol/10(7) plastids). Radiolabel incorporation into the P700 apoproteins, CP47 and CP43, saturated at a chlorophyll a concentration which corresponds to 50% of the etioplast protochlorophyllide content (0.06 nmol of chlorophyll a/10(7) plastids)

The proteome of the heterocyst cell wall in Anabaena sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

Abstract Methods for the cryopreservation of protein import and integration in pea chloroplasts and of protein import or protein synthesis in tobacco mitochondria were modified to yield enzymatically active cryopreserved etioplasts from barley (Hordeum vulgare L.). The cryoprotectants ethylene glycol and dimethy sulfoxide were about 64 and 77% effective, respectively, for the cryopreservation of etioplast intactness. Phototransformation of protochlorophyllide a, esterification of chlorophyllide a or zinc-pheophorbide a, and stabilization of the de novo synthesized plastid-encoded chlorophyll-apoproteins P700, CP47, CP43, D2, and D1 were successfully preserved in liquid nitrogen. Cryopreservation of freshly prepared intact etioplasts completely retained enzymatic activities for accumulation of chlorophyll a or resulted in a slightly decreased yield of zinc-pheophytin a.</jats:p

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.FAPESPCNP

Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b6f (Cytb6f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1-3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains

Choosing a control intervention for a randomised clinical trial

BACKGROUND: Randomised controlled clinical trials are performed to resolve uncertainty concerning comparator interventions. Appropriate acknowledgment of uncertainty enables the concurrent achievement of two goals : the acquisition of valuable scientific knowledge and an optimum treatment choice for the patient-participant. The ethical recruitment of patients requires the presence of clinical equipoise. This involves the appropriate choice of a control intervention, particularly when unapproved drugs or innovative interventions are being evaluated. DISCUSSION: We argue that the choice of a control intervention should be supported by a systematic review of the relevant literature and, where necessary, solicitation of the informed beliefs of clinical experts through formal surveys and publication of the proposed trial's protocol. SUMMARY: When clinical equipoise is present, physicians may confidently propose trial enrollment to their eligible patients as an act of therapeutic beneficence