A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.     

Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas

Photosystem I comprises 13 subunits in Chlamydomonas reinhardtii, four of which-the major reaction center I subunits PsaA and PsaB, PsaC and PsaJ-are chloroplast genome-encoded. We demonstrate that PSI biogenesis involves an assembly-governed regulation of synthesis of the major chloroplast-encoded subunits where the presence of PsaB is required to observe significant rates of PsaA synthesis and the presence of PsaA is required to observe significant rates of PsaC synthesis. Using chimeric genes expressed in the chloroplast, we show that these regulatory processes correspond to autoregulation of translation for PsaA and PsaC. The downregulation of translation occurs at some early stage since it arises from the interaction between unassembled PsaA and PsaC polypeptides and 5' untranslated regions of psaA and psaC mRNAs, respectively. These assembly-dependent autoregulations of translation represent two new instances of a control by epistasy of synthesis process that turns out to be a general feature of protein expression in the chloroplast of C. reinhardtii.     

Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly

The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is "search-and-capture," in which dynamically unstable microtubules (MTs) search space to capture chromosomes. Although existing theoretical estimates suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.     

Proteomic analysis of human placental syncytiotrophoblast microvesicles in preeclampsia

Background

Placental syncytiotrophoblast microvesicles (STBM) are shed into the maternal circulation during normal pregnancy. STBM circulate in significantly increased amounts in preeclampsia (PE) and are considered to be among contributors to the exaggerated proinflammatory, procoagulant state of PE. However, protein composition of STBM in normal pregnancy and PE remains unknown. We therefore sought to determine the protein components of STBM and whether STBM protein expressions differ in preeclamptic and normal pregnancies.Patients with PE (n = 3) and normal pregnant controls (n = 6) were recruited. STBM were prepared from placental explant culture supernatant. STBM proteins were analyzed by a combination of 1D Gel-LC-MS/MS. Protein expressions levels were quantified using spectral counts and validated by immunohistochemistry.

Results

Over 400 proteins were identified in the STBM samples. Among these, 25 proteins were found to be differentially expressed in preeclampsia compared to healthy pregnant controls, including integrins, annexins and histones.

Conclusion

STBM proteins include those that are implicated in immune response, coagulation, oxidative stress, apoptosis as well as lipid metabolism pathways. Differential protein expressions of STBM suggest their pathophysiological relevance in PE.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-40) contains supplementary material, which is available to authorized users.     

In vivo electron donation from plastocyanin and cytochrome c6 to PSI in Synechocystis sp. PCC6803

Many cyanobacteria species can use both plastocyanin and cytochrome c₆ as lumenal electron carriers to shuttle electrons from the cytochrome b₆f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast re-reduction of P₇₀₀ in vivo after a single charge separation. Here, we show that both cytochrome c₆ and plastocyanin can bind to PSI in the dark and participate to the fast phase of P₇₀₀ reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c₆ than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P₇₀₀ oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c₆, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.     

A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition.

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.     

Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.