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H Kuroiwa

Publications and source records attributed to H Kuroiwa.

At least 19 recordsLinked to original sources
PubMed2001

Pollen tube attraction by the synergid cell.

In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

Culture Techniques↗
PubMed2001

Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods.

Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO.

Animals↗
PubMed2001

Behavior of plastid nucleoids during male gametogenesis in Plumbago auriculata.

We characterized the behavior of plastid (pt) and mitochondrial (mt) nucleoids during male gametogenesis in Plumbago auriculata in three dimensions. The behavior of pt-nucleoids and mt-nucleoids differed throughout male gametogenesis. Pt-nucleoids were distributed in a characteristic manner in three stages: in the early microspore, pt-nucleoids assemble around cell nucleus; in the mid-generative cell, pt-nucleoids gather at the internal side of the pollen; in the late-generative cell, pt-nucleoids aggregation turns its pole to the external side of the pollen. We also studied organelle nucleoids in the egg and the central cell by a method in which semi-thick sections of resin-embedded anthers and ovaries were observed by confocal laser scanning microscopy. The number of pt-nucleoids in the sperm cell did not differ significantly from that in the egg. These results suggest that the behavior of DNA-containing organelles is regulated strictly during male gametogenesis in P. auriculata, and that a biparental inheritance of plastids in the Plumbago embryo is more favored than was previously thought.

Cell Nucleus↗
PubMed2001

The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae.

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings.

Carbocyanines↗
PubMed2001

Visualization of an FtsZ ring in chloroplasts of Lilium longiflorum leaves.

FtsZ is a bacterial division protein which forms a ring at the leading edge of the cell division site. To date, a hypothesis that the plant FtsZ forms the same structure in chloroplast division is proposed, but has not been demonstrated yet. In this study, recombinant LlFtsZ (Lilium longiflorum FtsZ) protein was produced from a previously isolated ftsZ cDNA clone [Mori and Tanaka (2000) Protoplasma 214: 57] and used to raise polyclonal anti-LlFtsZ antibodies in rabbits. In immunoblot analysis with the total protein extracted from L. longiflorum leaves, purified antibodies specifically recognized LlFtsZ whose molecular mass was approximately 43 kDa. This size corresponded to that of the recombinant LlFtsZ protein lacking N-terminal sequence, which suggests that the full-length LlFtsZ translation product has a putative N-terminal signal peptide. Moreover, fluorescent and electron microscopy revealed that the anti-LlFtsZ antibodies recognized ring structures at stromal side of the constriction point of dividing chloroplasts. Here, we show direct evidence that FtsZ ring is involved in chloroplast division.

Bacterial Proteins↗
PubMed2001

Plastid division is driven by a complex mechanism that involves differential transition of the bacterial and eukaryotic division rings.

During plastid division, two structures have been detected at the division site in separate analyses. The plastid-dividing ring can be detected by transmission electron microscopy as two (or three) electron-dense rings: an outer ring on the cytosolic face of the outer envelope, occasionally a middle ring in the intermembrane space, and an inner ring on the stromal face of the inner envelope. The FtsZ ring, which plays a central role in bacterial division, also is involved in plastid division and is believed to have descended to plastids from cyanobacterial endosymbiosis. The relationship between the two structures is not known, although there is discussion regarding whether they are identical. Biochemical and immunocytochemical investigations, using synchronized chloroplasts of the red alga Cyanidioschyzon merolae, showed that the plastid FtsZ ring is distinct and separable from the plastid-dividing ring. The FtsZ ring localizes in stroma and faces the inner plastid-dividing ring at the far side from the inner envelope. The FtsZ ring and the inner and outer plastid-dividing rings form in that order before plastid division. The FtsZ ring disappears at the late stage of constriction before dissociation of the plastid-dividing ring, when the constriction is still in progress. Our results suggest that the FtsZ ring;-based system, which originated from a plastid ancestor, cyanobacteria, and the plastid-dividing ring;-based system, which probably originated from host eukaryotic cells, form a complex and are involved in plastid division by distinct modes.

Arabidopsis Proteins↗
PubMed2000

Reversible thermal transition of soluble branched chains from slightly acid-treated potato starch.

The reversible thermal transition of soluble branched starch chains prepared from slightly acid-treated potato starch granules (ATS) was investigated. Potato starch was immersed in 15% sulfuric acid to obtain ATS with a 1% hydrolysis rate. About half of the molecules of ATS, which spontaneously formed large aggregates with a mass of a few million daltons in aqueous solution, was fractionated and soluble branched starch chains with a relative molecular weight (Mr) of 8.91 x 10(4) were obtained. Structural analysis indicated that the soluble branched starch chains consisted of three unit chains with Mr 7,900 and 21 unit chains with Mr 2,700. DSC and FT-IR measurements showed that the soluble branched starch chains underwent a reversible thermal transition, which is considered to be a helix-coil transition, during heating and cooling, but a debranched sample and beta-limit dextrins showed substantially different thermal behavior, indicating the contribution of the ordered structure of the branched chains.

Acids↗
PubMed1999

Pharmacokinetic analysis and antitumor efficacy of MKT-077, a novel antitumor agent.

MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [14C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of 14C after i.v. administration of [14C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of 14C decreased with a T1/2 of approximately 5 min. In the second and terminal phase, the T1/2 of 14C was 2.8-4.6 h and 16.2 h, respectively. Cmax (1 min after injection) increased from 0.3 to 1.5 microg/ml linearly, but less than proportionately between the doses. The AUC(0-infinity) at 0.3, 1 and 3 mg/kg were 0.030 +/- 0.002, 0.60 +/- 0.12 and 1.73 +/- 0.25 microg x h/ml, respectively. Plasma clearance was approximately 1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), 14C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), 14C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [14C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors.

Animals↗
PubMed1999

A pair of invertedly repeated genes in Chlamydomonas reinhardtii encodes a zygote-specific protein whose expression is UV-sensitive.

Uniparental inheritance of the chloroplast genome has been observed in a wide variety of green plants. In Chlamydomonas this phenomenon, which can be selectively inhibited by UV irradiation of mt(+) gametes, has been shown cytologically to be due to the preferential degradation of mt(-)-derived chloroplast nucleoids in young zygotes. The zygote-specific pair of zys1 genes, zys1A and zys1B, is expressed earliest among five genes isolated from a "10-min" zygote library. We report here that the ZYS1 protein, which is encoded by the invertedly duplicated zys1 gene, accumulates in zygotes and is localized in nuclei. In addition, when mt(+) gametes (but not mt(-) gametes) are UV-irradiated before mating, only very limited accumulation of ZYS1 protein can be detected in the resulting zygotes.

Algal Proteins↗
PubMed1999

The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance.

Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Hér, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole (DAPI) and 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type "m+p+", which has both mitochondrial and plastid DNA (P. zonale), (2) type "m+p-", which only has mitochondrial DNA (M. acuminata), (3) type "m-p+", which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type "m-p-", which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m- or p-). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI.

Cytoplasm↗
PubMed1999

Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga cyanidioschyzon merolae

In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions.

Journal Article↗
PubMed1999

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development.

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.

DNA, Mitochondrial↗
PubMed1999

Two ftsH-family genes encoded in the nuclear and chloroplast genomes of the primitive red alga Cyanidioschyzon merolae.

The red algal chloroplast genome encodes an essential prokaryotic cell division gene, ftsH, which has never been found in the mitochondrial genome of any organism. To compare the conserved prokaryote-derived mechanism for mitochondrial division with that of chloroplasts, we cloned chloroplast- and nuclear-encoded ftsH genes from the primitive red alga Cyanidioschyzon merolae. The deduced amino-acid sequence of chloroplast ftsH (ftsHcp) consists of 603 amino acids and shows the highest similarity with algal-chloroplast and cyanobacterial FtsH. On the other hand, the nuclear-encoded ftsH (ftsH2) encodes a protein of 920 amino acids and has the highest similarity with two yeast mitochondrial FtsHs, Rca1p and Afg3p. Furthermore, the amino-terminal extension of FtsH2 appears to be an amphipathic alpha-helix, a characteristic mitochondrial targeting signal, suggesting that FtsH2 is a mitochondrial protein. Southern hybridization revealed that ftsH2 is a single gene located on chromosome III of the 17 C. merolae chromosomes. The level of expression of the 3.0 and 4.0 kb transcripts of this gene decreased in concert during the organelle division phase of a synchronized culture, indicating a cell-cycle-dependent manner of ftsH2 transcription, while northern hybridization did not detect ftsHcp transcripts. Nevertheless, reverse transcription-PCR and immunoblotting demonstrated for the first time that chloroplast-encoded ftsH is transcriptionally and translationally active. Overproduction of FtsHcp and FtsH2 in Escherichia coli disrupted cytokinesis and produced filamentous cells, but had no effect on the replication, segregation, or distribution of their nucleoids, as also occurs in ftsH-deficient E. coli. These observations suggest the possible involvement of both C. merolae FtsHs in organelle division.

ATP-Dependent Proteases↗
PubMed1999

Characterization of Chlamydomonas reinhardtii zygote-specific cDNAs that encode novel proteins containing ankyrin repeats and WW domains.

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Amino Acid Sequence↗
PubMed1999

Improved sensitivity for high resolution in situ hybridization using resin extraction of methyl methacrylate embedded material.

An in situ hybridization procedure resulting in both high resolution and sensitivity was established by using the removable methyl methacrylate resin, Technovit 9100. Young bicellular pollen of tobacco (Nicotiana tabacum L. SR-1) was embedded in Technovit 9100 resin and sectioned. The resin was extracted with (2-methoxyethyl)-acetate followed by in situ hybridization with cRNA probes to detect cytoplasmic 18S/25S rRNA. Signal intensity obtained by this procedure was approximately twice as great as that obtained by an earlier procedure using Technovit 7100, a glycol methacrylate resin that cannot be removed from sections. This improvement in sensitivity made it possible to observe subcellular localization of small amounts of RNA as revealed by visualization of plastid 23S rRNA in a generative cell of Plumbago auriculata pollen.

Acetates↗
PubMed1998

Characterization of a chloroplast isoform of serine acetyltransferase from the thermo-acidiphilic red alga Cyanidioschyzon merolae.

We isolated a gene for serine acetyltransferase (SAT), a key enzyme in sulfate assimilation, from the primitive red alga Cyanidioschyzon merolae, an inhabitant of sulfurous hot springs, and designated this gene cmSAT. The N-terminal region of the cmSAT protein has characteristics of a chloroplast targeting peptide. cmSAT protein fused with a 6x histidine tag complemented a SAT deficient Escherichia coli mutant. The protein was purified with its SAT activity, which was inhibited by cysteine, using the high affinity of the histidine tag in an Ni-NTA column. The Km values for acetyl-CoA and l-serine were 0.3 and 0.1 mM, respectively. Southern blotting indicated the existence of other SAT isoforms in C. merolae. A 2.4 kb transcript was always detected when growth was synchronized under a 12-h light/dark cycle. Under these conditions, a 31-kDa protein was always detected on immunoblots, indicating processing of the cmSAT protein and constitutive expression of cmSAT. A 45-kDa protein, thought to be the unprocessed cmSAT protein, was detected in the dark period, from M phase to early G1 phase. No significant change in the level of protein expression was detected under continuous darkness or in a sulfate-deficient medium. Using immunoelectron microscopy, the cmSAT protein was primarily detected in the stroma and a few were detected in the cytoplasm, which indicate that cmSAT protein is transported to and functions in a chloroplast.

Acetyltransferases↗
PubMed1998

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles. Investigations of tissues and cells by fluorescence microscopy.

The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile.

Cotyledon↗