Checkpoint control on mitochondrial division inCyanidioschyzon merolae

Summary It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase , using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3-dihexyloxacarbocyanine iodide (DiOC₆; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.Abbreviation DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Mitochondrial division by an electron-dense ring inCyanidioschyzon merolae

Summary The first identification of a mitochondria-dividing ring (MD-ring), which is located in the cytoplasm near the outer envelope membrane at the constricted isthmus of dividing mitochondria in the red algaCyanidioschyzon merolae, is reported. The MD-ring is about 50 nm wide and 10 nm thick at early stage of mitochondrial constriction and is a somewhat electron-dense circular bundle. The MD-ring is believed to be essential for the division of mitochondrion (mitochondriokinesis) since the ring appears at the equatorial region of the mitochondria just before the initiation of mitochondrial division and can be observed throughout mitochondrial division. The MD-ring has features comparable to that of the plastid-dividing (PD) ring.Abbreviations MD mitochondria-dividing - PD plastid-dividing     

Isolation of the cell-nuclear,mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

Summary The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10³kbp, 1.6×10³kbp, and 5.0×10³kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.     

Close association of centrosomes to the distal ends of the microbody during its growth, division and partitioning in the green alga Klebsormidium flaccidum

Summary. Division and partitioning of microbodies (peroxisomes) of the green alga Klebsormidium flaccidum, whose cells contain a single microbody, were investigated by electron microscopy. In interphase, the rod-shaped microbody is present between the nucleus and the single chloroplast, oriented perpendicular to the pole-to-pole direction of the future spindle. A centriole pair associates with one distal end of the microbody. In prophase, the microbody changes not only in shape, from a rodlike to a branched form, but also in orientation, from perpendicular to parallel to the future pole-to-pole direction. Duplicated centriole pairs are localized in close proximity to both distal ends of the microbody. In metaphase, the elongated microbody flanks the open spindle, with both distal ends close to the centriole pair at either spindle pole. The microbody further elongates in telophase and divides after septum formation (cytokinesis) has started. The association between the centrioles and both distal ends of the microbody is maintained throughout mitosis, resulting in the distal ends of the elongated microbody being fixed at the cellular poles. This configuration of the microbody may be favorable for faithful transmission of the organelle during cell division. After cytokinesis is completed, the microbody reverts to the perpendicular orientation by changing its shape. Microtubules radiating from the centrosomes flank the side of the microbody throughout mitosis. The close association of centrosomes and microtubules with the microbody is discussed in respect to the partitioning of the microbody in this alga. Correspondence: H. Hashimoto, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan. Present address: M. Honda, Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.     

Orderly formation of the double ring structures for plastid and mitochondrial division in the unicellular red alga Cyanidioschyzon merolae

The formation of the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) was studied in a highly synchronous culture of the unicellular red alga Cyanidioschyzon merolae. The timing and the order of formation of the MD and PD rings were determined by observing organelles around the onset of their division, using transmission electron microscopy. In  C. merolae, there is one chloroplast and one mitochondrion per cell, and the shape of the chloroplast changes sequentially from acorn-like, to round, to trapezoidal, to peanut-shaped, in that order, during the early stage of chloroplast division. None of the cells with acorn-shaped or round chloroplasts contained organelles with PD rings or MD rings, while all of the cells with peanut-shaped chloroplasts contained organelles with both PD rings and MD rings. In cells with peanut-shaped chloroplasts, the PD and MD rings were double ring structures, with an outer ring located on the cytoplasmic face of the outer membrane of the organelle, and an inner ring located in the matrix beneath the inner membrane. These results suggested that the double ring structures of the PD ring and the MD ring form when chloroplasts are trapezoidal in shape. Detailed three-dimensional observation of cells with trapezoidal chloroplasts revealed the following steps in the formation of the double ring structures of the PD and MD rings: (i) the inner ring of the PD ring forms first, followed by the outer ring; (ii) then the MD ring forms and becomes visible; (iii) when the double ring structures of the two rings have formed, the microbody then moves from its remote location to the plane of division of the mitochondrion and contraction of the PD and MD rings commences. These steps were also confirmed by computer-aided three-dimensional reconstruction of the images from serial thin sections. This study reveals the order of formation of the double ring structures of the PD and MD rings, and the behavior of the microbody around the onset of division of plastids and mitochondria. The results also provide the first evidence that the inner PD ring is not a tension element formed by the contractile pressure but a definite structure, independent of the outer ring. Received: 31 March 1998 / Accepted: 14 May 1998     

Biosynthesis of a microbody matrix enzyme in greening cotyledons

Earlier work on microbody biosynthesis has shown that glyoxysomal and liver peroxisomal proteins synthesized in the cytosol are made as precursors which are then transferred into the organelles and processed. Here, it is demonstrated that the unprecessed precursor detected in the cytosol after protein synthesis in vivo for an enzyme at the transition stage between glyoxysomes and leaf peroxisomes is indistinguishable from the product of translation in vitro. It is assumed that the transfer of extraorganellarly made precursor across the glyoxysomal membranes is followed by processing of the precursor and oligomerization to the tetrameric or 16-meric form of the enzyme. Oligomerization was, however, also observed in a portion of the cytosolic form.     

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

The taxonomic position of Cyanidium,Cyanidioschyzon and Galdieria: an update

The ecophysiological, cytomorphological, biochemical and molecular data presently available for the acidophilic red algal species Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are summarised. The taxonomic position of the three genera is discussed and emendements to the generic diagnosis are presented.     

Pityrialactone- a new fluorochrome from the tryptophan metabolism of M. alassezia furfur

As the main nitrogen source in Malassezia (M.) furfur, tryptophan induces the formation of fluorochromes and pigments, which makes the yeast less sensitive towards UV light. For the investigation of the fluorochromes, M. furfur (CBS1878) was incubated at 32 °C for 14 days on a pigment-inducing medium, and the agar extract was purified by column chromatography, preparative TLC and HPLC. The structures of the pure metabolites were determined by mass spectrometry and NMR spectroscopy. A pale yellow compound eluting from the column with 22% acetonitrile was found to exhibit a strong green-yellow fluorescence. The fluorochrome is a new bisindolyl compound (C₂₀H₁₂N₂O₃, MW 328.33) named pityrialactone because of its furan-2,3-dione structure. The UV protective properties (λ_max 352, 292, 276, 224 nm) of this metabolite were confirmed in a yeast model. As shown by the fluorescence spectrum, pityrialactone appears to be responsible for the green-yellow fluorescence of pityriasis versicolor lesions under Wood light. Pityrialactone is accompanied by the isomeric bisindolylmaleic anhydride (pityriaanhydride), which has not yet been described as a natural product but is a known intermediate in the total synthesis of bisindolylmaleimides. This revised version was published online in August 2006 with corrections to the Cover Date.     

Localization changes of endogenous hydrogen peroxide during cell division cycle of Xanthomonas

Production and localization of endogenous hydrogen peroxide (H₂O₂) were investigated in strains of Xanthomonas by histochemical analysis under electron microscopy. Even though the levels of endogenous H₂O₂ production were different among various strains, the produced H₂O₂ was localized in the cell wall of all Xanthomonas strains tested. The impairment of the level of endogenous H₂O₂ accumulation resulted in a significantly decreased growth rate of bacteria, regardless if the difference of the H₂O₂ level is originally present between wild type strains or caused by mutation of the ahpC gene of Xanthomonas. The endogenous accumulation of H₂O₂ positively correlates with the cell division. Interestingly, the accumulated H₂O₂ was also localized in the mesosome-like structure and nucleoids during the cell division cycle. Furthermore, results revealed quantitative and dimensional changes of H₂O₂ accumulation in the two additional locations. These findings indicated that the additional locations of the accumulated H₂O₂ were closely associated with the process of cell division. Together, these results suggest that the endogenous H₂O₂ production plays an important role in cell proliferation of Xanthomonas.     

The centennial of the Fenton reaction

A short account is given of Fenton's life and research, with special emphasis on the Fenton reactions.     

Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae

As part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H₂O₂ by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH^· by growing the bacteria at acidic pH, or in the presence of an OH^· scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host.     

Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae

The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.     

Evolutionary Analysis of the Small Heat Shock Proteins in Five Complete Algal Genomes

Small heat shock proteins (sHSPs) are chaperones that are crucial in the heat shock response but also have important nonstress roles within the cell. sHSPs are found in all three domains of life (Bacteria, Archaea, and Eukarya). These proteins are particularly diverse within land plants and the evolutionary origin of the land plant sHSP families is still an open question. Here we describe the identification of 17 small sHSPs from the complete genome sequences of five diverse algae: Chlamydomonas reinhardtii, Cyanidioschyzon merolae, Ostreococcus lucimarinus, Ostreococcus tauri, and Thalassiosira pseudonana. Our analysis indicates that the number and diversity of algal sHSPs are not correlated with adaptation to extreme conditions. While all of the algal sHSPs identified are members of this large and important superfamily, none of these sHSPs are members of the diverse land plant sHSP families. The evolutionary relationships among the algal sHSPs and homologues from bacteria and other eukaryotes are consistent with the hypothesis that the land plant chloroplast and mitochondrion sHSPs did not originate from the endosymbionts of the chloroplast and mitochondria. In addition the evolutionary history of the sHSPs is very different from that of the HSP70s. Finally, our analysis of the algal sHSPs sequences in light of the known sHSP crystal structures and functional data suggests that the sHSPs possess considerable structural and functional diversity. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Rüdiger Cerff     

Generation of hydroxyl radicals by soybean nodule leghaemoglobin

Leghaemoglobin, a protein present in root nodules of soybean (Glycine max (L.) Merr.), generates the highly reactive hydroxyl radical (·OH) upon incubation with hydrogen peroxide (H₂O₂). The H₂O₂ appears to cause breakdown of the haem, releasing iron ions that convert H₂O₂ into ·OH outside the protein. Oxyleghaemoglobin (oxygenated ferrous protein) is more sensitive to attack by H₂O₂ than is metleghaemoglobin (ferric protein). The possibility of oxyleghaemoglobin breakdown by H₂O₂ and formation of damaging ·OH may explain why the root nodule is equipped with iron-storage proteins and enzymes that can remove H₂O₂.     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

Cell division across the volume of colonies of flavobacterium sp. 22

Six-day-old colonies ofFlavobacterium sp. 22 were studied by electron microscopy. Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth ofFlavobacterium sp. 22 is not purely peripheral. It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface. For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.     

Catecholamine-containing nerve terminals of the eccrine sweat glands of macaques

Summary A loose network of catecholamine-containing nerves was demonstrated with a fluorescence histochemical method (Falck-Hillarp) in the coiled portion of eccrine sweat glands in the digital pads of macaques after the injection of nialamide and noradrenaline. In the skin of untreated control animals, fluorescent fibers appear only in some of the glands. A systemic administration of reserpine and a local injection of 6-hydroxydopamine (6-OHDA) or 5-hydroxydopamine (5-OHDA) into the digital pad cause a complete disappearance of fluorescent fibers around the glands and blood vessels. Electron micrographs reveal many unmyelinated varicose axon profiles outside the basement membrane of secretory tubules. Most of these profiles contain many small agranular vesicles and a few large dense-cored vesicles (cholinergic terminal), and some have numerous small granular and a few large densecored vesicles (adrenergic terminal).The local injection of 6-OHDA causes various degenerative changes in the adrenergic terminals but the cholinergic ones and the rest of the cellular structure remain intact. The injection of 5-OHDA induces a significant increase of electron-dense granules in the vesicles of adrenergic terminals.The presence of catecholamine and the effects of 6-OHDA and 5-OHDA in the nerve terminals indicate that the innervation of the eccrine sweat glands of macaques consists of cholinergic as well as adrenergic terminals.Publication No. 783 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grant RR 00163 of the Animal Resources Branch, Division of Research Resources.We acknowledge the excellent assistance of Tsutomu Yoshida, Tsuneka zu Fuse, John Ochsner, and Nickolas Roman.     

耐辐射奇球菌pprM基因增强大肠杆菌氧化抗性的研究

目的:探讨耐辐射奇球菌ppr M基因对大肠杆菌氧化抗性的影响。方法:氯化钙法转化分别构建含pGEX-6p-1-pprM质粒的大肠杆菌DH5α。测定不同浓度过氧化氢对含pGEX-6p-1-pprM、pGEX-6p-1和野生型大肠杆菌DH5α活性的影响以及菌体内SOD/GSH/CAT水平的变化。结果:与空质粒组和野生型组相比,含pprM的大肠杆菌在同浓度过氧化性情况下,其抑菌圈明显缩小,差异有统计学意义。与空质粒组和野生型组相比,含pprM的大肠杆菌体内CAT活力、SOD活性明显提高,但GSH量并没有明显提高。结论:pprM基因能够提高大肠杆菌抗氧化能力,其机制可能与pprM基因增强细菌体内抗氧化酶的活性有关。