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We describe a boy with Fisher syndrome. He presented the typical symptoms of Fisher syndrome, including external ophthalmoplegia, abnormality of convergence, and areflexia, after an episode of Campylobacter enterocolitis. Atypically, however, anti-GA1 antibody was detected in his serum, though anti-GQ1b and anti-GT1a antibodies were not. In addition, the tau protein level in his cerebrospinal fluid was elevated. Generally, Fisher syndrome is a self-limiting disease and has a good prognosis. In our patient, however, mild diplopia and areflexia persisted 6 months after their onset. Here, we report on the first Fisher syndrome patient with anti-GA1 antibody in the serum and elevated tau protein in the cerebrospinal fluid.

We report two individual cases of cardio-facio-cutaneous (CFC) syndrome with severe neurological impairment consisting of infantile spasms with hypsarrhythmia and refractory epilepsy with multifocal epileptic paroxysms such as modified hypsarrhythmia. Both cases shared diffuse brain atrophy and severely delayed myelination on neuroimaging. Genetic analysis revealed individual heterozygous mutations in the KRAS (phenotype of CFC/Noonan syndrome) and BRAF genes (phenotype of CFC syndrome). Neurological impairment in cases with mutations in the RAS/MAPK (mitogen activated protein kinase) signal pathway may be more severe, and could be linked to some forms of refractory epilepsy, especially epileptic encephalopathy that includes infantile spasms.

Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

SUMMARY A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 108 cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.

We report three familial cases of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis syndrome, including a pair of monozygotic twins and their mother. It suggests that periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis syndrome may have a certain monogenetic background.

本研究の対象とする島根県東部の平野部では，過去の被害地震において震源から離れているにもかかわらず，被害が生じた地域であることが報告されている．その原因として地盤震動特性，地盤速度構造が少なからず影響したものと考えられる．そこで出雲平野および松江平野において微動アレイ観測および重力異常データに基づき推定された地盤構造について検討を行った．微動データによるS波速度構造と重力データによる2次元解析結果と比較すると，S波速度2000～2500m/s層までの深度と近い値を示すことが分かった．この結果を踏まえて3次元解析を行い，面的に基盤構造を把握することができた．

浜田市では1872年浜田地震(M7.1)の際，甚大な被害が発生したとの記録があり，特に市街地で被害が甚大であったとされる．そこで本研究では，浜田市街地において４地点の微動アレイ観測の実施ならびに既往の研究で得られている単点３成分観測記録を用いて地盤構造の推定を行った．微動アレイ観測記録の解析から，Ｓ波速度100～3200m/sの地下構造モデルを推定できた．浜田市内の沖積層のS波速度は100～150ｍ/sであった．H/Vのピークは明瞭な単峰型が多く，その周期は0.2～0.8秒で浜田川沿いの市街地で周期が長くなることがわかった．S波速度構造，H/Vの特徴から軟弱な沖積層が市内全域に広く分布していることがわかった．基盤深度については，基盤岩(Vs=800m/s層)までの深さは最深部で約50mであり，地震基盤相当の最下層（Vs=3000m/s相当層）までの深さは，最大で約800mであることがわかった．

In the Uranouchi Inlet, which has a commercial fishery of littleneck clam Ruditapes philippinarum, potentially toxigenic species of Dinophysis acuminata showed an annual occurrence except for the summer of 2000–2002. Relatively high abundances of D. acuminata were observed in April 2000 and November 2002. Some samples of D. acuminata cells, which were collected from the inlet in autumn 2002, contained pectenotoxin-2 (0.9–2.8 pg/cell) and okadaic acid (1.9–2.8 pg/cell). Cell length and hypotheca width of D. acuminata found in the Uranouchi Inlet were 33.7–45.0 and 21.0–31.6 μm, respectively. The length-width ratios ranged 1.21–1.71. Sequences of the 5.8S ribosomal RNA gene and flanking internal transcribed spacers, 1 and 2 of cells of D. acuminata from the inlet fell into a D. acuminata complex clade that included, D. acuminata-D. sacculus-D. acuta reported by others. In the inlet, occurrences of D. acuminata were detected within a broad water temperature range between 13.0 and 26.9°C, and those showing more than 25 cells/L were within 16.4–17.3°C for temperature and 27.6 and 34.1 for salinity. This is the first report that cells of Dinophysis acuminata from southern Japanese coastal waters contained okadaic acid and pectenotoxin-2.

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R>0.92 (p

The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

We evaluated the suitability of ELF-based assay (ELF: Enzyme-Labeled Fluorescence, Molecular Probes) as a tool for specific-detection of alkaline phosphatase (AP) activity in individual species of marine phytoplankton. Among 4 species of marine phytoplankton tested in the present culture experiments, the ELF-labeled cells of Karenia mikimotoi (Dinophyceae) and Skeletonema costatum (Bacillariophyceae) were simultaneously found with AP activity that was determined by using 4-methylumbelliferyl phos- phate under the phosphorus-starved state but not found under the phosphorus-replete state. There were significant positive correlations between the percentage of ELF-labeled cells and AP activity of K. mikimotoi and of S. costatum. In contrast for Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae) which do not produce AP, no ELF-labeled cells were found even under the phosphorus- starved state. Thus, ELF-based assay is considered to be useful for specific-detection of AP activity.

Utilization of phosphate diester (PDE) and phosphodiesterase (PDEase) production by 5 marine phytoplankton s pecies were examined in the laboratory to evaluate the contribution of PDE to the growth of phytoplankton. Among the 5 marine phytoplankton species tested, only Chaetoceros ceratosporus was able to use the PDE compound (bis(p-nitrophenyl) phosphate) effectively as a sole phosphorus source. In addition, C. ceratosporus simultaneously produced both PDEase and alkaline phosphatase (APase) at almost equal activity-levels under the phosphate-deficient condition. These results suggest that PDE compounds presumably play an important role as a phosphorus source for PDEase-producing phytoplankton in coastal environments. Many studies have generally focused on dissolved inorganic phosphate (orthophosphate: PO4 3- ) as the major phosphorus source for the growth of phytoplankton to evaluate the factors controlling occurrences of algal blooms (Nalewajko and Lean, 1980). However, the

沿岸海水中のアルカリフォスファターゼ (AP) 活性とフォスファターゼ加水分解性リン (APHP) の分布と消長を解析した。浦ノ内湾における海水中のAP活性は高水温期に高く, プランクトン態AP活性の寄与が大きいこと, 本湾ならびに野見湾, 浦戸湾および広島湾海水中には, 0.02未満～0.29μMのAPHPが溶存しており, 海域による有意な濃度差は無いことがわかった。これらのことから, 沿岸海水中のAPHPは植物プランクトンのリン源として有効に利用されることが示唆された。

赤潮原因種Karenia mikimotoi およびSkeletonema costatum はリン酸モノエステル利用能とアルカリフォスファターゼ (AP) 産生能を有し, Heterosigma akashiwo はこれらの能力を欠くことが明らかになった。前2種のAP活性はそれぞれ最大115および0.273fmol/cell/minであり, いずれも活性は極めて高いことがわかった。これらのことから, 海水中の有機態リンが赤潮の発生に対して有意に寄与していることが示唆された。

We designed five 16S rRNA-targeted oligonucleotide probes (Sp probes) specific for Flavobacterium sp. 5 N-3, which inhibits the growth of red tide phytoplankton Gymnodinium mikimotoi (Dinophyceae). These probes were evaluated by whole-cell hybridization against 5 N-3 cells incubated under laboratory conditions. The fluorescence signal from the cell detected with Sp probe mix5, a mixture of the five probes, was 8.4-fold higher than that obtained with only one Sp probe (Sp01RF). The signal obtained by this method was strong enough to recognize 5 N-3 cells and count them under the epifluorescence microscope, while the signal was often undetectable when a single probe was used. Fluorescence intensities of cells at stationary phases and of ‘starved’ cells in sterile seawater using Sp probe mix5 were low but still sufficient for enumeration. These Sp probes did not hybridize with 11 strains from the Cytophaga/Flavobacteria/Bacteroides phylum and did react with strain 5 N-3 following whole-cell hybridization. These results show that 5 N-3 cells cultivated under our laboratory conditions can be detected by whole-cell hybridization with the five designed probes. These data also suggest that this technique may be useful for detection of an algicidal bacterium 5 N-3 in the natural environment.

The relationship between the abundance of the toxic marine dinoflagellate Alexan- drium tamarense (Lebour) Balech and cyst formation-inhibiting bacteria (Alex-CFIB) was investi- gated in samples taken from the water column in Hiroshima Bay (Japan) in 1999. The cell density of A. tamarense peaked in the middle of April and blooms declined in May. Alex-CFIB were detected during the bloom period as well as the non-bloom period in 1999 by means of the most probable number (MPN) bioassay as well as the colony counting method. A total of 32 strains that had poten- tial Alexandrium cyst formation-inhibiting activities (CFIB) were isolated from the seawater samples from Hiroshima Bay throughout the year. The population structure and genetic diversity of Alex- CFIB were analyzed by means of restriction fragment length polymorphism (RFLP) of the 16S ribo- somal RNA genes (16S rDNA). Five ribotypes, Ia to Id and II types, were determined among the 32 strains of Alex-CFIB. Most of the strains belonged to ribotype I, suggesting that bacteria of ribotype I may be dominant in the Alex-CFIB assemblages in the field seawater. Almost the entire 16S rDNA- based phylogenetic tree showed that ribotypes I and II fell into the class Proteobacteria γ-subdivision Alteromonas group and theVibrio group, respectively. The 6-well microplate approach clarified that Alex-CFIB obtained in this study do not have growth-inhibiting activities, and Alex-CFIB of ribotype I (Alteromonas group) have strong activities of encystment inhibition among these ribotypes. The existence not only of Alexandrium cyst formation-promoting bacteria (Alex-CFPB) reported previ- ously but also of Alex-CFIB in Hiroshima Bay throughout the year suggests that Alex-CFPB, as well as Alex-CFIB, especially bacteria of ribotype I, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment.

Microdeletion of 16q12 is a rare chromosomal abnormality. We present the cases of two Japanese patients with developmental and renal symptoms of differing clinical severity. Both patients had 16q12 interstitial microdeletions that included the entire SALL1 gene. Patient 1 was a 15-year-old Japanese boy clinically diagnosed with branchio-oto-renal syndrome with mild developmental delay, but with no imperforate anus or polydactyly. Array comparative genome hybridization (aCGH) indicated a 5.2 Mb deletion in 16q12, which included SALL1. Patient 2 was a 13-year-old Japanese boy diagnosed with Townes-Brocks syndrome and severe developmental delay, epilepsy, and renal insufficiency requiring renal replacement therapy. Fluorescence in situ hybridization indicated deletion of the entire SALL1 gene. Subsequent aCGH showed a 6 Mb deletion in 16q12q13, which included SALL1. Precise analysis of the present two cases will give us some clues to elucidate the pathogenic mechanisms of 16q12 microdeletion syndrome.

The relationship between the abundance of the toxic marine dinoflagellate Alexandrium tamarense (Lebour) Balech and Alexandrium-cyst-formation-promoting bacteria (Alex-CFPB) was investigated in the water column of Hiroshima Bay (Japan) from 1997 to 1998. Cell density of A. tamarense increased gradually from February to the middle of April, then peaked at the end of April and blooms declined rapidly in the beginning of May in both years. All seawater fractions collected from 5 m depth, where the density of A. tamarense cells was highest and which also contained the bulk of planktonic bacteria, promoted cyst formation of A. catenella (Whedon and Kofoid) Balech. This promotion was not caused by effects from nutrient limitation. The number of Alex-CFPB in seawater samples, analyzed by means of the most probable number (MPN) method, increased from the beginning of the Alexandrium bloom and reached 3.60 x 10 3 and 1.00 x 10 3 cells ml -1 at the peak bloom period at the end of April in 1997 and 1998, respectively. As the blooms declined, the number of Alex-CFPB decreased rapidly to less than 10 cells ml-1. Alexandrium-cyst-formation-inhibiting bacteria (Alex-CFIB) were not detected. These results show a clear positive correlation between the abundance of A. tamarense and Alex-CFPB during blooms, which suggests that Alex-CFPB play a significant role in the process of encystment and bloom dynamics of Alexandrium in the field.

This report describes a simplified method for direct counting of total bacteria associated with the fleshy red algae Kappaphycus alvarezii (Doty) Doty and Gracilaria spp. A Nuclepore® polycarbonate membrane (0.2–μm pore size) fitted to a vacuum filtration apparatus was used to filter algal tissue homogenate after serial dilution and staining with the fluorochrome 4′,6-diamidino-2-phenylindole. Using epifluorescence microscopy, it is possible to count bacteria without preseparating them from the algae. The technique requires homogenized algal tissue diluted with 0.2-μm-filtered, autoclaved seawater to a level appropriate for counting. Dilution reduces the amount of autofluorescent algal debris, which may interfere with Counting. The membrane filtration method yielded a bacterial count two orders of magnitude higher than that of the conventional agarspread plate technique. This method offers a more accurate approach to counting the total number of bacteria on macroalgae.

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4', 0a, 6'', 6c, ?s, 5''', 0p, and 2''''. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2'''' plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3'' plate and the rectangular shaped 2' plate.

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.

The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS-1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS-1 (WKS-I type) or A. tamarense CU-15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS-1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (

BackgroundThe marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.Methods/principal findingA total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006-2011. The SSU rDNA, the LSU rDNA D8-D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.Conclusions/significanceThis study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8-D10 as genetic markers are recommended for further use.

We studied the effects and tolerability of lamotrigine (LTG) on refractory epilepsies in persons with severe motor and intellectual disabilities (SMID).We introduced LTG to 40 subjects all who were severely retarded and suffered from symptomatic epilepsies consisting of six cases of generalized epilepsy (GE), 33 of partial epilepsy (PE), and one of unclassified epilepsy (according to the 1989 ILAE classification standards). We evaluated the efficacy of LTG in seizure reduction and its tolerability for persons with SMID.Following treatment, 83% of GE and 42% of PE cases showed a 50% reduction in seizure frequency. From the seizures observed in our study cohort, a response rate (RR) with an over 50% reduction was found in 54% of secondarily generalized tonic-clonic (n = 24), 30% of complex partial (n = 18), and 25% of myoclonic seizures (n = 16). As for adverse effects, nine subjects (22.5%) experienced sleep problems, six subjects (15%) experienced excitement mainly during night-time, and four cases each (10%) experienced involuntary movements and sleepiness. As for additional effects on patients' quality of life (QOL), even in poor responder (50% RR) patients, LTG improved "alertness" in six cases, improved "activity of daily life" in four cases, and helped to diminish "appetite loss" in three cases.LTG therapy for epileptic conditions in persons with SMID has potential for not only reducing seizure frequency, but also for improving QOL.

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.

This study was conducted with a particular focus on preterm infants with West syndrome (WS) to evaluate differences in the first responses to oral medication based on etiology.Medical records of 53 patients with WS, treated at five institutions between 2005 and 2009, were reviewed retrospectively. Patients were divided into six groups based on the time of brain insult, and evaluated for short-term outcomes using oral anti-epileptic agents and synthetic adrenocorticotropic hormone.The sample consisted of 15, six, 14, two, four, and 12 patients classified, on the basis of apparent time of acquisition of etiology, into the prenatal, term, preterm, postnatal, other, and no identified etiology groups, respectively. Average age of onset in the term group was 3.3 ± 1.0 months, significantly earlier than in the prenatal, preterm, postnatal and no identified etiology groups (P0.05). All patients in the term group had experienced seizures before the onset of WS. Only patients in the preterm group had only experienced neonatal seizures, and responded better to treatment. Patients in the preterm group had better responses to treatment, especially oral medication, compared with those in the prenatal and term groups. The prevalence of relapse of seizures in the preterm group (14%) was significantly lower than that in the prenatal group.Preterm WS patients responded well to treatment. Distinguishing WS patients on the basis of different etiologies is important for evaluating the effectiveness of treatment.

Eight isolates of the toxic dinoflagellate Alexandrium tamarense from four different bays of Japan and the Gulf of Thailand were examined for differentiation using seven monoclonal antibodies, M8751-1, M151-1, M22-1, M102-3, MT15-1, MT15-2, and MT15-4, against cell surface antigens of Alexandrium.Antibodies M8751-1, M102-3, and M151-1 derived from Japanese strains reacted to all the Japanese strains of A. tamarense tested, but not to Thai strains by means of indirect immunofluorescence assay, while two antibodies MT15-2 and MT15-4 derived from the Thai strain reacted to two Thai strains but not to all Japanese strains. Antibodies M22-1 and MT15-1 reacted with all strains from both countries. These data corresponded with the results of ELISA and SDSPAGE analysis of whole protein. Using the western-blotting method, M8751-1 reacted with a protein band at about 50kDa, which were found in most of the Japanese isolates, but not in the Thai ones.All isolates that have been morphologically identified as the same species, A. tamarense, are grouped into two different populations according to monoclonal antibodies. One of these is the Japanese population, while the other group is the Thai one. These monoclonal antibodies appear to be useful for the identification of intra-and inter-species of these organisms.

Eight monoclonal antibodies reactive against cultured cell surface antigens of genus Alexandrium (Dinophyceae) were tested for cross-reaction with natural field samples from Japanese coastal waters, by the indirect immunofluorescence (IIF) method.In advance of applying monoclonal antibodies to field samples, the reactivity of typical antibodies to cultured cells at various growth stages in asexual and sexual cycles were examined by dot immunoassay and IIF, respectively. As a result, the reactive intensities were constant during the exponential growth phase and decreased a little during the stationary phase. At a different stage of sexual reproduction, these antibodies reacted with the vegetative cells of parent and F1 progenies, but not with the planozygotes and cysts. The reactive spectra of these antibodies to the cells of natural field samples collected from Tanabe Bay (Wakayama Pref.) in May 1990 and 1991, from Ofunato Bay (Iwate Pref.) in June 1991, and from Tachibana Bay (Nagasaki Pref.) in July 1991 almost coincided with those of these antibodies to cultured cells of A. catenella, A. tamarense, and A. affine, respectively. The results of immunological analysis corresponded with those of the morphological features.These monoclonal antibodies appear to be useful for the identification of these organisms from natural sea water.

Heterotrophic bacteria isolated from water samples taken from Hiroshima Bay, Japan, and referred to as Alexandrium (Dinophyceae) cyst formation-promoting bacteria, were assigned to the Roseobacter–Sulfitobacter–Silicibacter group within the α-Proteobacteria on the basis of nearly complete 16S rRNA gene sequences. Phylogenetic analyses showed that two strains, CFPB-A9T and CFPB-A5, are closely related to each other and that their closest relative was Jannaschia helgolandensis (95·9 % sequence similarity). These strains were Gram-negative, motile, obligately aerobic rods that required sodium ions and 2–7 % sea salts for growth and did not produce bacteriochlorophyll a. Their optimal growth temperature was 25–30 °C. The strains had Q-10 as the dominant respiratory quinone. Primary cellular fatty acid in both strains was 18 : 1ω7c. The DNA G+C contents of strains CFPB-A9T and CFPB-A5 were 59·1 and 59·2 mol%, respectively. Based on physiological, biological, chemotaxonomic and phylogenetic data, the strains are considered to represent a novel species, Jannaschia cystaugens sp. nov., with type strain CFPB-A9T (=LMG 22015T=NBRC 100362T).

A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense . The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA). Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, Mbo I, Hha I, Rsa I and Bst UI. Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A. tamarense bloom in 1998, belonged to ribotype A. This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods. The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the α subclass of the Proteobacteria . Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup. The non- Rhodobacter group type fell into the Marinobacterium - Neptunomonas - Pseudomonas group of the γ- Proteobacteria . Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions. These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs. These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment.

We diagnosed Pallister-Mosaic syndrome (PMS) in a 4-month-old female infant. In addition to the presence of non-specific anomalies, involving anorectal, finger and ear anomalies, characteristic cranio-facial features and irregular skin lesions that appeared after age 2 months suggested the possibility of genetic mosaicism, PMS in particular. Fluorescence in situ hybridization technique revealed an extra copy of chromosome 12p; i (12p) in 30% of cultured skin fibroblasts. When focal skin lesions accompany neurodevelopmental disabilities in early infancy, genetic analysis for mosaicism should be considered for differential diagnosis. Significantly, we describe several phenotypic features and neuroimaging findings of the PMS in the present case, which have not been described in previous reports. The neuroimaging abnormalities we encountered, such as polymicrogyria, speculating congenital brain anomaly, may explain the severe motor and intellectual disabilities of PMS.

Caspases are believed to play a key role in the delayed neuronal cell death observed in the rat brain after hypoxic-ischemic (HI) insult. Caspase inhibitors have been developed as antiapoptotic agents. Hippocampal damage after HI insult is strongly related to tissue temperature, and systemic hypothermia has been introduced clinically for brain protection. In this study, we examined the effects of a caspase inhibitor and systemic hypothermia on neuronal protection in the developing rat brain. Postnatal d 7 rat pups were subjected to the Rice model of hypoxia for 1 h. Systemic hypothermia was induced with a water bath at 29 degrees C. Before HI insult, a pan-caspase inhibitor, boc-aspartyl-(OMe)-fluoromethyl-ketone (BAF), was injected into the cerebral ventricle. The ipsilateral hippocampus was subjected to caspase assays and histologic assessment. The HI group at 37 degrees C (HI-37 degrees C) showed a peak of caspase-3 activity 16 h after insult. This activity was significantly reduced in the presence of BAF or hypothermia (HI-29 degrees C group, p < 0.05) or by the combination of HI-29 degrees C + BAF (p < 0.01 versus HI-37 degrees C). The number of neuronal cells in the ipsilateral hippocampal CA1 region in the HI-37 degrees C group was significantly decreased (62.9% versus control). The number of neuronal cells was maintained in the HI-37 degrees C + BAF group (82.7%), the HI-29 degrees C group (78.7%), and the combination group (95.2%) (p < 0.05 versus HI-37 degrees C). A combination of systemic hypothermia and BAF produced a strong protective effect against neuronal damage in the developing rat brain, along with a reduction in caspase-3 activity.

BACKGROUND: A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. METHODS/PRINCIPAL FINDING: In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. CONCLUSIONS/SIGNIFICANCE: This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

The internal oxidation of iron alloys containing between 0.069 and 0.274 wt pct aluminum was investigated in the temperature range from 1223 to 1373 K for the purpose of determining the diffusion coefficients in γ-iron as well as in the internal oxidation layer. A parabolic rate law is obeyed in the internal oxidation of the present alloys. The rate constant for penetration of the oxidation front, the oxide formed, and the concentration of aluminum in the oxidation layer were determined. Pronounced enrichment of aluminum in the oxidation layer was observed, resulting from the counterdiffusion of aluminum. The oxygen concentration at the specimen surface was determined by combining the thermodynamic data on the dissociation of FeO and the solution of oxygen in y-iron. The diffusion coefficient of oxygen in the internal oxidation layer,D o 10 , was evaluated on the basis of the rate equation for internal oxidation.D o 10 increases at a given temperature as the volume fraction of oxide,f 10, in the oxidation layer increases. The diffusion coefficient of oxygen in γ-iron,D o, was determined by extrapolation ofD o 10 = 0.D o may be expressed as $$D_o = \left( {1.30\begin{array}{*{20}c} { + 0.80} \\ { - 0.50} \\ \end{array} } \right) \times 10^{ - 4} \exp \left[ { - \frac{{166 \pm 5(kJ \cdot mol^{ - 1} )}}{{RT}}} \right]m^2 \cdot s^{ - 1} .$$ D o is close to the diffusion coefficients of carbon and nitrogen in γ-iron.