Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.The most essential pigment-protein complexes for oxygenic photosynthesis are PSI and PSII, which are embedded in the thylakoid membranes of chloroplasts and cyanobacteria. Cooperation between PSI and PSII achieves light-driven noncyclic electron transport from the oxidative splitting of water to the reduction of ferredoxin and is accompanied by the generation of a proton gradient for ATP synthesis. Phycobilisomes (PBS), another pigment-protein complex, are attached to the stromal side of the thylakoid membrane in cyanobacteria and red algae; they work as light-harvesting antennae to transfer electronic excitation energy mainly to PSII and, in some cases, to PSI (Gantt 1994). The integration of these pigment-protein complexes changes in response to light conditions, nutrient status, and developmental stage (Fujita et al., 1994; Grossman et al., 1994; Wolk et al., 1994).Some cyanobacteria, including Anabaena variabilis, are able to grow diazotrophically using the nitrogen-fixing enzyme nitrogenase. Because nitrogenase is sensitive to oxygen, oxygenic photosynthesis is not readily compatible with diazotrophic growth. When this filamentous cyanobacterium is grown under fixed nitrogen-deficient conditions, approximately 1 in 10 to 20 vegetative cells differentiates into a heterocyst, in which oxygenic photosynthesis is suppressed and nitrogenase becomes operative (Haselkorn, 1978; Wolk et al., 1994). The other vegetative cells continue oxygenic photosynthesis. The differentiation of heterocysts from chains of vegetative cells has been studied extensively (Golden and Yoon, 2003; Toyoshima et al., 2010). The abundances of PSII and PBS decrease during the transition. PSI appears to persist in the heterocyst to produce ATP by cyclic electron transport, because nitrogen fixation demands a large amount of ATP (Wolk et al., 1994). However, the mechanisms by which PBS and PSII are degraded during heterocyst differentiation remain unclear, and whether the amount of PSI per cell changes is unknown.The PBS of A. variabilis contain three types of phycobiliproteins, pigment-protein complexes with distinct absorption and fluorescence spectra. The core PBS contains allophycocyanin (APC), which absorbs around 654 nm (Ying and Xie, 1998); the core is most closely connected to PSII. More peripherally in the PBS, the so-called rod contains phycoerythrocyanin (PEC) and phycocyanin (PC), which absorb maximally around 575 and 604 to 620 nm, respectively (Switalski and Sauer, 1984; Zhang et al., 1998). Photon energy is absorbed by PEC, then transferred downhill through PC and APC and finally to PSII. The structure of PBS is probably optimized not only for efficient energy transfer to PSII and/or PSI but also for transformation and/or degradation under various nutrient conditions. However, the order in which these subunits degrade during heterocyst differentiation remains unknown. One strategy to address this question is to isolate heterocysts at several stages during differentiation and quantify their proteomes via mass spectrometry. However, such isolation procedures work well only when there is a good understanding of the properties of cells at different stages. Ideally, noninvasive methods should be used to understand changes in the integrity of PSII and PBS in intact cells in filaments.In principle, time-lapse microscopic observations can clarify the process of differentiation from a vegetative cell into a mature heterocyst. Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al., 1981; Ying et al., 2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007; Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz and Montgomery, 2010; Collins et al., 2012, Sugiura and Itoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments. However, to date, there have been no thorough time-lapse investigations of the fluorescence spectra of heterocysts and vegetative cells during the differentiation process.In this study, we investigated the dynamic changes in thylakoid membranes of A. variabilis during heterocyst differentiation. Our unique microscopic system can acquire fluorescence spectra from an entire linearly illuminated region with about 2-nm wavelength resolution in a single exposure (Kumazaki et al., 2007). Heterocyst formation was induced by transferring vegetative cell filaments from fixed-nitrogen-sufficient incubation medium to nitrogen-deprived medium. We conducted long-term observations (60–96 h) on identical filaments. Another unique feature of our setup is that it uses a near-infrared (NIR) excitation laser source. Our previous microspectroscopic study of chloroplasts of a higher plant, maize (Zea mays), and a green alga (Parachlorella kessleri) showed that continuous wave (CW) laser light emitting at 785 to 820 nm excited PSI with high selectivity under the one-photon excitation (OPE) mode. This enabled us to observe highly PSI-rich fluorescence spectra and images with signals around 710 to 740 nm, even at approximately 295 K (Hasegawa et al., 2010, 2011). We used this technique to quantify PSI in individual heterocysts compared with its parental and contiguous vegetative cells. Pigment fluorescence under OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typically achieved with picosecond or femtosecond pulses), because TPE using 800 to 830 nm resulted in spectra with contributions from PBS, PSII, and PSI, as typically observed by visible light excitation (Kumazaki et al., 2007; Hasegawa et al., 2010, 2011). The advantages of our microscopic system are the high wavelength resolution and coverage of the entire fluorescence spectrum, the availability of fluorescence spectra at several differentiation stages, and the multiple excitation modes with different selectivities for pigment-protein complexes. Together, these analyses allowed us to characterize spectral decomposition and to understand the time dependence of different pigment-protein complexes, even at a physiological temperature. Microscopic absorption spectra were also obtained from single cells. These data were tentatively used to estimate the absolute concentrations of PSI and PSII in heterocysts and vegetative cells.     

Evaluation of antioxidant activity of leafy vegetables and beans with myoglobin method

Key message

Antioxidant activity of seven leafy vegetables and four beans against five reactive oxygen species and reactive nitrogen species was clearly characterized with a protocol using myoglobin as a reporter probe.

Abstract

Antioxidant activity of seven leafy vegetables and four beans against peroxyl radical, hydroxyl radical, hypochlorite ion, and peroxynitrite ion has been measured using myoglobin as a reporter probe (myoglobin method). Conventional DPPH method was also used to evaluate antioxidant activity of the samples. Difference of activity against different reactive oxygen species (ROS) and reactive nitrogen species (RNS) was characterized by plotting the data in a 5-axe cobweb chart. This plot clearly showed the characteristics of the antioxidant activity of the leafy vegetables and the beans. The samples examined in this work were categorized into four groups. (1) The samples showed high antioxidant activity against all ROS and RNS: daikon sprout, spinach, Qing-geng-cai, and onion. (2) The samples showed high antioxidant activity against peroxyl radical: red bean and soy bean. (3) The samples showed high antioxidant against hypochlorite ion: broccoli floret, cabbage, and Chinese cabbage. (4) The samples showed weak antioxidant activity against all ROS and RNS: cowpea and common beans. Our protocol is probably useful to characterize antioxidant activity of the crops of different cultivars, the crops obtained in different growing environments and growing seasons, the crops harvested at different age, and the crops stored in the different conditions, as well as the changes of activity during cooking process of the crops.     

Orally administered <Emphasis Type="Italic">Bifidobacterium</Emphasis> triggers immune responses following capture by CD11c<Superscript>+</Superscript> cells in Peyer’s patches and cecal patches

We have investigated the immunomodulatory mechanisms of Bifidobacterium pseudocatenulatum JCM7041 (Bp) as model of probiotics following oral administration to mice. This study was conducted with the aim of clarifying the mechanism of immunomodulation induced by oral administration of probiotic bacteria through elucidation of the detailed mechanism of transfer of orally administered bacterial cells within the body and the interaction between bacterial cells and cells of the immune tissues. We observed the localization of Bp in mice following oral administration, showing that Bp was surrounded by CD11c⁺ cells in Peyer’s patches (PP) and cecal patches (CP). These results indicated that Bp might induce CD11c⁺ cell-mediated immune responses directly. Furthermore, IL-10 and IL-12p40 production by Thy1.2⁻ cells, including CD11c⁺ cells, increased significantly. Production of IL-10 and IL-12p40 by bone marrow-derived dendritic cells (BMDC) was significantly increased by Bp stimulation. These results suggest that oral administration of Bp induces immune responses directly following capture by CD11c⁺ dendritic cells (DCs). Subsequently, we observed oral administration of Bp for 1 week induced IgA and IgA-associated cytokine production by CP and PP cells, suggesting that Bp induced DC-mediated immune responses on CP as well as PP.     

Molecular evolution of immunoglobulin superfamily genes in primates

Genes of the immunoglobulin superfamily (IgSF) have a wide variety of cellular activities. In this study, we investigated molecular evolution of IgSF genes in primates by comparing orthologous sequences of 249 IgSF genes among human, chimpanzee, orangutan, rhesus macaque, and common marmoset. To evaluate the non-synonymous/synonymous substitution ratio (ω), we applied Bn-Bs program and PAML program. IgSF genes were classified into 11 functional categories based on the Gene Ontology (GO) database. Among them, IgSF genes in three functional categories, immune system process (GO:0002376), defense response (GO:0006952), and multi-organism process (GO:0051704), which are tightly linked to the regulation of immune system had much higher values of ω than genes in the other GO categories. In addition, we estimated the average values of ω for each primate lineage. Although each primate lineage had comparable average values of ω, the human lineage showed the lowest ω value for the immune-related genes. Furthermore, 11 IgSF genes, SIGLEC5, SLAMF6, CD33, CD3E, CEACAM8, CD3G, FCER1A, CD48, CD4, TIM4, and FCGR2A, were implied to have been under positive selective pressure during the course of primate evolution. Further sequence analyses of CD3E and CD3G from 23 primate species suggested that the Ig domains of CD3E and CD3G underwent the positive Darwinian selection.     

Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.     

WRN counteracts the NHEJ pathway upon camptothecin exposure

We investigated the function of the interaction between WRN (Werner syndrome gene product) and Ku70 and between WRN and DNA-PKcs, which are components of the DNA-PKcs/Ku70/Ku80 complex, by generating KU70(-/-)/WRN(-/-) and DNA-PKcs(-/-/-)/WRN(-/-) double-gene knockout chicken DT40 cells. When treated with camptothecin (CPT), an inhibitor of DNA topoisomerase I, WRN(-/-) cells showed higher sensitivity than wild-type cells, whereas KU70(-/-) and DNA-PKcs(-/-/-) cells showed hyper-resistance. Disruption of KU70 or DNA-PKcs suppressed the sensitivity of WRN(-/-) cells to CPT, rendering them as resistant to CPT treatment as KU70(-/-) and DNA-PKcs(-/-/-) cells. On the other hand, CPT sensitivity of BLM(-/-) cells, which are defective in a RecQ helicase similar to WRN, was enhanced by deletion of KU70. The implications for the function of WRN in the non-homologous end-joining pathway of DNA repair involving Ku70 and DNA-PKcs, which may be the cause of lethality in the presence of CPT, will be discussed.     

Genetic basis of rheumatoid arthritis: A current review

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. As with other complex traits, genome-wide association studies (GWASs) have tremendously enhanced our understanding of the complex etiology of RA. In this review, we describe the genetic architecture of RA as determined through GWASs and meta-analyses. In addition, we discuss the pathologic mechanism of the disease by examining the combined findings of genetic and functional studies of individual RA-associated genes, including HLA-DRB1, PADI4, PTPN22, TNFAIP3, STAT4, and CCR6. Moreover, we briefly examine the potential use of genetic data in clinical practice in RA treatment, which represents a challenge in medical genetics in the post-GWAS era.     

Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation

DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other''s enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape.