Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris

Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.     

IDENTIFICATION OF THE MINUS MATING‐TYPE SPECIFIC GENE MTD1 FROM GONIUM PECTORALE (VOLVOCALES,CHLOROPHYTA)1

Gonium pectorale O. F. Müll. (Volvocales, Chlorophyta), a colonial 8‐ or 16‐cellular alga, is phylogenetically important as an intermediate form between isogametic unicellular Chlamydomonas and oogamous Volvox. We identified the mating‐type specific gene GpMTD1, from G. pectorale, the first homologue of Chlamydomonas reinhardtii MTD1 (CrMTD1). The GpMTD1 gene was found to be present only in the minus mating‐type locus and was expressed specifically in the gametic phase as is the case for CrMTD1, suggested to participate in development of the minus gametes. This gene is useful as a probe in analyzing the bacterial artificial chromosome (BAC) library for resolving genomic structures of the mating‐type loci in isogamous and oogamous colonial volvocaleans.     

Further discussion about the features of Lake Puma Yum Co, South Tibet, China

Further discussion about the limnological features of Lake Puma Yum Co, South Tibet, China, is provided based on the results of several investigations. By using depth data from all over the lake, the whole submarine topography has been compiled. Horizontal analysis of the water's physicochemical features indicates that compared with the relatively uniform water features at other lake areas, apparent spatial heterogeneity exists in the water of the subaquatic alluvial fan induced by the Jiaqu River, the biggest inflow. Vertical analysis of water characteristics using two-factor analysis of variance with no re-experiment indicates that temperature, dissolved oxygen, and pH of the water vary with water depth rhythmically, whereas other parameters demonstrate no evident vertical variation, which shows that chemical stratification is not obvious. But this does not exclude slightly higher concentrations of Ca²⁺ induced by lower pH at the bottom of deep lake water. The hydrochemistry difference between inflow water and lake water reveals the loss of Ca²⁺ in lake water, which indicates calcite deposition may be an important characteristic of lake sediment.     

Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales,Chlorophyceae)

A strain of Chloromonas pseudoplatyrhyncha (Pascher) P. C. Silva, which has not been studied previously using cultured material, was established from a soil sample collected in Japan and examined by light microscopy, transmission electron microscopy, and molecular phylogenetic analyses. The chloroplasts of this species showed no pyrenoids under light microscopy. However, transmission electron microscopy and the staining methods with carmine after fixation in an acidified hypochlorite solution revealed that Chloromonas pseudoplatyrhyncha actually had multiple, atypical pyrenoids (pyrenoid matrices without associated starch grains) that were angular in shape and distributed in the interior regions of the lobes of the chloroplasts. Although some other species of Chloromonas have atypical pyrenoids in the chloroplast, such angular pyrenoids have not previously been reported within the Volvocales. The present molecular phylogenetic analysis, based on 18S ribosomal RNA, adenosine triphosphate synthase β‐subunit, and P700 chlorophyll a‐apoprotein A2 gene sequences, demonstrated that Chloromonas pseudoplatyrhyncha belonged to the Chloromonas lineage or Chloromonadinia, in which it occupied a basal position outside a robust, large monophyletic group consisting of 13 species of Chloromonas and Gloeomonas.     

Tubulin polymerization inhibitors with a fluorinated phthalimide skeleton derived from thalidomide

4,7-Difluoro-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione [4,7FPP-33 (14)] has a potent tubulin-polymerization-inhibiting activity comparable with those of the known tubulin-polymerization inhibitors rhizoxin and colchicine. The structure-activity relationship for fluorine substitution was elucidated.     

Angiotensin II type 1 (AT1)-receptor blocker prevents impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

Our aim was to test for smoking-induced endothelial dysfunction in rat cerebral vessels, then to evaluate the effect of valsartan [angiotensin II type I (AT1)-receptor blocker] on that impairment. In pentobarbital-anesthetized, mechanically ventilated Sprague-Dawley rats, we used a cranial window preparation to measure changes in pial vessel diameters following topical applications of acetylcholine (Ach) (before and after smoking or intravenous nicotine infusion; n = 6 in each group), and adenosine (n = 6 for before and after smoking). Then, after intravenous valsartan pretreatment we reexamined the pial vasodilator response to topical Ach (before and after cigarette smoking). Under control conditions, cerebral arterioles were dilated by 6.9 +/- 4.2% and 13.6 +/- 4.8% by topical Ach (10(-6) M and 10(-5) M, respectively) and by 6.4 +/- 2.5% and 12.2 +/- 3.1% by topical adenosine (10(-5) M and 10(-4) M, respectively). One hour after a 1-min inhalation of mainstream smoke (1-mg nicotine cigarette), 10(-5) M Ach constricted cerebral arterioles (-4.4 +/- 4.1%), while 10(-4) M adenosine dilated them by 13.4 +/- 3.4%. One hour after a 1-min nicotine infusion (0.05 mg), 10(-5) M Ach dilated cerebral arterioles by 9.9 +/- 2.4%. Thus, vasodilator response to topical Ach was impaired after smoking, whereas that to adenosine was unaffected. However, the vasodilator response to Ach was unaffected by intravenous nicotine. Valsartan prevented smoking from impairing Ach-induced vasodilation. In conclusion, acute single-cigarette smoking causes a dysfunction of endothelium-dependent, but not endothelium-independent, vasodilation of rat cerebral vessels in vivo, and the effect was not mimicked by intravenous nicotine. AT1-receptor blockade prevented the above smoking-induced impairment of endothelium-dependent vasodilation.     

AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS1

Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat‐based dimorphism of chloroplast DNA (cpDNA). Here, we examined the “shallow” and “deep” subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima’s D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both “shallow” and “deep” haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat‐based dimorphism of cpDNA from field‐collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.