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To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1— but not SKOR, AKT1, and SAG14—were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

Although attempts to improve the phosphate (Pi) uptake and use efficiency by constitutively overexpressing phosphate transporters have resulted in enhanced Pi or total phosphorous contents, growth promotion by Pi acquisition was observed in only a few cases. This study examined the effect of the tissue-specific overexpression of phosphate transporter on Pi acquisition and plant growth. We cloned cDNA for a wheat phosphate transporter, TaPT2, using PCR and confirmed its Pi transport activity in Arabidopsis suspension cells. The overexpression of TaPT2 by the Arabidopsis Shaker family inward rectifying potassium channel 1 (AKT1) promoter, dominantly expressed in root epidermal cells, resulted in increased root and shoot growth of transgenic Arabidopsis under Pi-replete and Pi-depleted conditions. However, their Pi and total P contents did not increase. The overexpression of TaPT2 by the constitutive promoter, actin8 (ACT8), increased shoot total P contents in transgenic plants, but did not promote their growth. These results suggested that enhanced Pi uptake in root epidermal cells is suitable as a driving force for Pi transport from roots to shoots, improving subsequent Pi use in shoots. Thus, the root epidermal cell-specific expression of TaPT2 may be a simple and promising strategy for enhancing plant Pi uptake and efficiency.

Class II high-affinity potassium transporters (HKT2s) mediate Na+–K+ cotransport and Na+/K+ homeostasis under K+-starved or saline conditions. Their functions have been studied in yeast and X. laevis oocytes; however, little is known about their respective properties in plant cells. In this study, we characterized the Na+ and K+ transport properties of SvHKT2;1, SvHKT2;2 and HvHKT2;1 in Arabidopsis under different ionic conditions. The differences were detected in shoot K+ accumulation and root K+ uptake under salt stress conditions, K+ accumulation in roots and phloem sap under K+-starved conditions, and shoot and root Na+ accumulation under K+-starved conditions among the HKT2s transgenic lines and WT plants. These results indicate the diverse ionic transport properties of these HKT2s in plant cells, which could not be detected using yeast or X. laevis oocytes. Furthermore, Arabidopsis expressing HKT2s showed reduced salt tolerance, while over-expression of HvHKT2;1 in barley, which has the ability to sequestrate Na+, showed enhanced salt tolerance by accumulating Na+ in the shoots. These results suggest that the coordinated enhancement of Na+ accumulation and sequestration mechanisms in shoots could be a promising strategy to confer salt tolerance to glycophytes.

Class II high-affinity potassium transporters (HKTs) have been proposed to mediate Na+-K+ co-transport in plants, as well as Na+ and K+ homeostasis under K+-starved and saline environments. We identified class II HKTs, namely SvHKT2;1 and SvHKT2;2 (SvHKTs), from the halophytic turf grass, Sporobolus virginicus. SvHKT2;2 expression in S. virginicus was up-regulated by NaCl treatment, while SvHKT2;1 expression was assumed to be up-regulated by K+ starvation and down-regulated by NaCl treatment. Localization analysis revealed SvHKTs predominantly targeted the plasma membrane. SvHKTs complemented K+ uptake deficiency in mutant yeast, and showed both inward and outward K+ and Na+ transport activity in Xenopus laevis oocytes. When constitutively expressed in Arabidopsis, SvHKTs mediated K+ and Na+ accumulation in shoots under K+-starved conditions, and the K+ concentration in xylem saps of transformants was also higher than in those of wild-type plants. These results suggest transporter-enhanced K+ and Na+ uploading to the xylem from xylem parenchyma cells. Together, our data demonstrate that SvHKTs mediate both outward and inward K+ and Na+ transport in X. laevis oocytes, and possibly in plant and yeast cells, depending on the ionic conditions.

The Arabidopsis high-affinity K+ transporter (AtHKT1;1) plays roles in salt tolerance by unloading Na+ from the root xylem to the xylem parenchyma cells and/or uploading Na+ from the shoot/leaf xylem to the xylem parenchyma cells. To use this promoter for the molecular breeding of salt-tolerant plants, I evaluated the expression profile of the AtHKT1;1 promoter in detail. Approximately 1.1 kbp of sequence upstream from the start codon of AtHKT1;1 was polymerase chain reaction (PCR)-amplified, fused to the β-glucuronidase (GUS) gene, and introduced into Arabidopsis. The resultant transformants were evaluated under nonstressed and salt-stress conditions at the seedling and reproductive stages. Histochemical analysis showed that GUS activity was detected in vascular bundle tissue in roots, hypocotyls, petioles, leaves, and petals, and in root tips. GUS enzyme activity in shoots tended to be higher than that in roots at both stages. After treatment with 50 mM NaCl for 24 h, GUS transcription levels and GUS enzyme activity were enhanced in transgenic lines. These results indicate that the AtHKT1;1 promoter isolated in this study could be useful in expressing transgenes specifically in vascular tissue and root tips, and in a mild salt-stress-responsive manner. The data provide novel insights into the functions of AtHKT1;1.

We characterized an Na+ transporter SvHKT1;1 from a halophytic turf grass, Sporobolus virginicus. SvHKT1;1 mediated inward and outward Na+ transport in Xenopus laevis oocytes and did not complement K+ transporter-defective mutant yeast. SvHKT1;1 did not complement athkt1;1 mutant Arabidopsis, suggesting its distinguishable function from other typical HKT1 transporters. The transcript was abundant in the shoots compared with the roots in S. virginicus and was upregulated by severe salt stress (500 mM NaCl), but not by lower stress. SvHKT1;1-expressing Arabidopsis lines showed higher shoot Na+ concentrations and lower salt tolerance than wild type (WT) plants under nonstress and salt stress conditions and showed higher Na+ uptake rate in roots at the early stage of salt treatment. These results suggested that constitutive expression of SvHKT1;1 enhanced Na+ uptake in root epidermal cells, followed by increased Na+ transport to shoots, which led to reduced salt tolerance. However, Na+ concentrations in phloem sap of the SvHKT1;1 lines were higher than those in WT plants under salt stress. Based on this result, together with the induction of the SvHKT1;1 transcription under high salinity stress, it was suggested that SvHKT1;1 plays a role in preventing excess shoot Na+ accumulation in S. virginicus.

Aegilops tauschii is the diploid progenitor of the bread wheat D-genome. It originated from Iran and is a source of abiotic stress tolerance genes. However, little is known about the molecular events of salinity tolerance in Ae. tauschii. This study investigates the leaf transcriptional changes associated with long-term salt stress. Total RNA extracted from leaf tissues of control and salt-treated samples was sequenced using the Illumina technology, and more than 98 million high-quality reads were assembled into 255,446 unigenes with an average length of 1398 bp and an N50 of 2269 bp. Functional annotation of the unigenes showed that 93,742 (36.69%) had at least a significant BLAST hit in the SwissProt database, while 174,079 (68.14%) showed significant similarity to proteins in the NCBI nr database. Differential expression analysis identified 4506 salt stress-responsive unigenes. Bioinformatic analysis of the differentially expressed unigenes (DEUs) revealed a number of biological processes and pathways involved in the establishment of ion homeostasis, signaling processes, carbohydrate metabolism, and post-translational modifications. Fine regulation of starch and sucrose content may be important features involved in salt tolerance in Ae. tauschii. Moreover, 82% of DEUs mapped to the D-subgenome, including known QTL for salt tolerance, and these DEUs showed similar salt stress responses in other accessions of Ae. tauschii. These results could provide fundamental insight into the regulatory process underlying salt tolerance in Ae. tauschii and wheat and facilitate identification of genes involved in their salt tolerance mechanisms.

Simple sequence repeat (SSR) markers are highly informative and widely used in genetic studies and plant breeding. Sporobolus virginicus is a halophytic turf grass that shows a high tolerance to salinity of up to 1.5 M NaCl. In the present study, we developed 148,411 SSR markers on 48,512 transcriptome contigs derived from RNA sequencing data. Of 33 randomly selected SSR markers, 23 (69.7%) produced clean amplification products, and an average of 1.25 alleles per marker was detected in S. virginicus genotypes. These markers were also examined by detecting polymorphisms across 19 different genera/species/genotypes in Poaceae, resulting in a high percentage (40%) of transferability. The sequencing of amplified products from these genera/species/genotypes revealed a high level of sequence similarity; however, substitutions, deletions, and insertions were detected not only in the objective SSRs but also in their flanking sequences. This is the first study on SSR marker development from S. virginicus. These SSR markers are useful for mapping the genes in and the breeding of S. virginicus and also for comparative genomics across genera/species/genotypes in Poaceae.

We produced transgenic Arabidopsis plants that express chimeric genes for transcription factors converted to dominant repressors, using Chimeric REpressor gene-Silencing Technology (CRES-T), and evaluated the salt tolerance of each line. The seeds of the CRES-T lines for ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23 exhibited higher germination rates than Wild type (WT) and developed rosette plants under up to 200 mM NaCl or 400 mM mannitol. WT plants did not grow under these conditions. In these CRES-T lines, the expression patterns of stress-related genes such as RD29A, RD22, DREB1A, and P5CS differed from those in WT plants, suggesting the involvement of the six transcription factors identified here in the stress response pathways regulated by the products of these stress-related genes. Our results demonstrate additional proof that CRES-T is a superior tool for revealing the function of transcription factors.

Our previous study showed that acute restraint stress enhances depolarization-induced increases in intrasynaptosomal free calcium (Ca(2+)) concentration ([Ca(2+)](i)) and Ca(2+)-dependent glutamate release in mouse cerebrocortical nerve terminals (synaptosomes). In the present study, we investigated the effects of chronic stress on [Ca(2+)](i) and glutamate release in cerebrocortical synaptosomes from mice.Male ddY strain mice were randomly assigned to one of two experimental groups: control group and chronic stressed group. Mice in the chronic stressed group were subjected to immobilization stress for 2 hours daily for a period of 21 days. [Ca(2+)](i) and glutamate release in cerebrocortical synaptosomes isolated from the mice were determined by fura-2 fluorescence assay and enzyme-linked fluorometric assay, respectively.Chronic stress caused a significant increase in resting [Ca(2+)](i) and significantly enhanced the ability of the depolarizing agents K(+) and 4-aminopyridine (4-AP) to increase [Ca(2+)](i). It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K(+)- and 4-AP-evoked Ca(2+)-dependent glutamate release. Synaptosomes were more sensitive to the depolarizing agents at lower concentrations following chronic stress than after acute stress. The pretreatment of synaptosomes with a combination of omega-agatoxin IVA (a P-type Ca(2+) channel blocker) and omega-conotoxin GVIA (an N-type Ca(2+) channel blocker) completely suppressed the enhancements of [Ca(2+)](i) and Ca(2+)-dependent glutamate release in chronic stressed mice.These results indicate that chronic stress enhances depolarization-evoked glutamate release by increasing [Ca(2+)](i) via stimulation of Ca(2+) entry through P- and N-type Ca(2+) channels, and that chronic stress increases the sensitivity to depolarizing agents.

To clarify the bolt bearing effect to the ultimate strength of the high-strength bolt connections, two series of testing, bolted connection applied with tension force or bending moment, were conducted in this study. From the test results, design formula, considering the bearing deformation explicitly, was proposed to evaluate the ultimate tension strength of the high-strength bolt connection; the improved accuracy compared with current design formula was also shown. Extending the proposed design formula to the bolted connection applied with bending moment, improved accuracy to predict the maximum bending strength was also shown; however, it was also reminded some refinement still needed to improve the accuracy., Transactions of AIJ. Journal of structural and construction engineering

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is an enzyme involved in formaldehyde metabolism in eukaryotes. FALDH cDNA was cloned from golden pothos, which is reported to effectively purify gaseous formaldehyde from enclosed room atmosphere. FALDH cDNAs from Arabidopsis, rice and golden pothos were overexpressed in transgenic Arabidopsis, and the enzyme activity was compared to determine the one most suitable for the molecular breeding of formaldehyde-detoxifying plants. The transgenic lines exhibited modified levels of the FALDH transcript, i.e. 10-800% compared to the endogenous transcript, due to either an overexpression or a cosuppression phenotype. The enzyme activity in the crude leaf extract was not proportionate, but did correlate with the transcription levels with certain exceptions. The FALDHs from the three plant species indicated similar enzymatic activity on average. The capacity to detoxify exogenous formaldehyde in the transformants with the FALDHs was determined at the whole plant level. Plants overexpressing FALDH from the three plant species displayed up to a 40% increase in their efficiency to take up exogenous formaldehyde as compared with the wild-type plants. On the other hand, no difference in the survival rate was observed among the transformants and wild type plants on formaldehyde-containing agar medium. These results show the FALDH from golden pothos to be similarly or more effective for detoxifying formaldehyde in transgenic plants compared with Arabidopsis and rice, and that this cDNA is applicable to the molecular breeding of formaldehyde detoxifying plants.

We have previously reported that expression of salt-responsive genes, including Bruguiera gymnorhiza ankyrin repeat protein 1 (BgARP1), enhances salt tolerance in both Agrobacterium tumefaciens and Arabidopsis. In this report, we further characterized BgARP1-expressing Arabidopsis to elucidate the role of BgARP1 in salt tolerance. BgARP1-expressing plants exhibited more vigorous growth than wild-type plants on MS plates containing 125–175 mM NaCl. Real-time PCR analysis showed enhanced induction of osmotin34 in the 2-week-old transformants under 125 mM NaCl. It was also showed that induction of typical salt-responsive genes, including RD29A, RD29B, and RD22, was blunted and delayed in the 4-week-old transformants during 24 h after 200 mM NaCl treatment. Ion content analysis showed that transgenic plants contained more K+, Ca2+, and NO3 −, and less NH4 +, than wild-type plants grown in 200 mM NaCl. Our results suggest that BgARP1-expressing plants may reduce salt stress by up-regulating osmotin34 gene expression and maintaining K+ homeostasis and regulating Ca2+ content. These results indicate that BgARP1 is functional on a heterogeneous background.

To identify key genes in the regulation of salt tolerance in the mangrove plant Bruguiera gymnorhiza, transcriptome profiling in the lateral and main roots under conditions of salt stress was performed. Statistical analysis revealed that 175 and 403 of 11,997 genes shoewd significantly increased high expression in the lateral and main roots respectively. One hundred and sixty genes were up-regulated in both types of roots in the early time period, 1 to 12 h after salt treatment. Expression vectors for 28 selected salt responsive genes were constructed and transformed in Agrobacterium tumefaciens, and then screened for salt tolerance. A. tumefaciens transformed with genes for lipid transfer, zinc finger, and ankyrin repeat proteins showed enhanced salt tolerance. Transgenic Arabidopsis plants expressing these three genes also exhibited increased tolerance to NaCl. These results indicate that Agrobacterium functional screening is an effective supplemental method of pre-screening genes involved in abiotic stress tolerance.

Summary To identify key genes in the regulation of salt tolerance in the mangrove plant Bruguiera gymnorhiza , cDNA expression libraries were constructed from salt-treated roots and leaves using the host organism Agrobacterium tumefaciens . Functional screening of the Agrobacterium libraries identified 44 putative salt tolerance genes in B.gymnorhiza . A cDNA clone which is homologous to an unknown cDNA from the mangrove plant K. candel and the cyc02 gene from Catharanthus roseus conferred the highest level of salt tolerance (450 mM NaCl) to A. tumefaciens , indicating that it plays a major role in the regulation of salt tolerance in mangrove plants. Several of the genes that we identified have not previously been implicated in plant salt tolerance. Transgenic Arabidopsis plants expressing Bg70 and cyc02 homologue exhibited increased tolerance to NaCl. These results demonstrate that Agrobacterium functional screening is an effective supplemental method to pre-screen genes involved in abiotic stress tolerance.

To identify key proteins in the regulation of salt tolerance in the mangrove plant Bruguiera gymnorhiza, proteome analysis of samples grown under conditions of salt stress was performed. Comparative two-dimensional electrophoresis revealed that two, three and one protein were differentially expressed in the main root, lateral root and leaf, respectively, in response to salt stress. Among these, three proteins were identified by internal peptide sequence analysis: fructose-1,6-bisphosphate (FBP) aldolase and a novel protein in the main root, and osmotin in the lateral root. These results suggest that FBP aldolase and osmotin play roles in salt tolerance mechanisms common to both glycophytes and mangrove plants. Osmotin was abundant at early time points following salt treatment and seems to play a role in initial osmotic adaptation in lateral roots of B. gymnorhiza under salt stress, but does not contribute towards adaptation to prolonged or continuous exposure to salt stress. The amounts of these proteins were not correlated with those of the respective mRNAs, as determined by microarray analysis. A novel salt-responsive protein, not previously detected by expressed sequence tag analysis or transcriptome analysis, was also identified in this proteomic approach, and may provide insight into the salt tolerance mechanism of the mangrove plant. This is the first report of proteome analysis with detailed analysis of main and lateral roots of mangrove plants under salt stress conditions.

A 61-year-old woman was admitted for head injury after a traffic accident. Two months later, she developed abducens nerve palsy, chemosis, and pulsatile tinnitus. Right internal carotid angiography demonstrated a high flow direct carotid-cavernous fistula (CCF) at the C(5) portion with reflux into the superficial and deep sylvian veins, superior ophthalmic vein, superior petrosal sinus, and inferior petrosal sinus. Intravascular ultrasonography (IVUS) revealed a large fistula at the C(5) portion of the internal carotid artery (ICA). Coil embolization via transarterial and transvenous approaches under IVUS monitoring was performed. During the procedure, IVUS accurately detected protrusion of a coil into the parent ICA, and the parent artery could be preserved. IVUS monitoring is useful for embolization of direct CCF with coils.

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.

We investigated the transcriptional response of Burma mangrove (Bruguiera gymnorhiza) to high salinity (salt stress; 500 mM NaCl) and hyperosmotic stress (osmotic stress; 1 M sorbitol) by microarray analysis. ANOVA (P < 0.05) and significant analysis of microarray (SAM; FDR < 5%) revealed that 865 of 11,997 genes showed significant differential expression under salt and osmotic stress. Scatter plot analysis revealed that the expression level of genes changed at 6 h after salt stress treatment, but recovered at 24 h, while the change at 6 h after osmotic stress treatment diverged at 24 h. Hierarchical clustering of the 865 genes showed that expression profiles under salt stress were distinctly different from those under osmotic stress. Comparison of gene ontology (GO) categories of differentially expressed genes under the stress conditions revealed that the adaptation of Burma mangrove to salt stress was accompanied by the up-regulation of genes categorized for “cell communication,” “signal transduction,” “lipid metabolic process,” “photosynthesis,” “multicellular organismal development,” and “transport,” and by down-regulation of genes categorized for “catabolic process.” Burma mangrove maintained its leaf water potential and recovered from its photosynthesis rate that declined temporarily under salt stress, but not under osmotic stress. These results demonstrated a fundamental difference between the response to salt and osmotic stress. Ion and sugar content analysis suggested that salt tolerance of Burma mangrove might be attributed to their ability to accumulate high concentrations of Na+ and Cl−, even under non-stressed conditions; to uptake additional Na+ and Cl− for use as osmolytes; and to maintain K+ homeostasis under salt stress.

Genes involved in the fertility restoration (Rf) of wild-abortive (WA)-type cytoplasmic male sterility (CMS) in rice have not been clearly identified, because of the inconsistency of their number and map position. Although the Rf-1 genes, which restore Chinsurah Boro (BT)-type CMS, have been recently isolated, their effects on WA-type CMS are still unclear. In the present study, near-isogenic lines (NILs) of japonica rice, differing at Rf loci, were developed using marker-assisted selection (MAS) to determine the effect of each Rf gene on WAor BT-cytoplasm in a japonica background. The NILs were crossed with a japonica CMS line, then the pollen and seed fertility of the hybrid plants was analyzed. The Rf-1 locus consisting of two Rf genes from IR36 was sufficient to restore fertility in the hybrids with BT-type CMS but conferred very little fertility to those with WA-type CMS. Rf-3 on chromosome 1, a weak Rf gene, and Rf-6(t) on chromosome 10 were unable to confer seed fertility to plants with BT-type or WA-type CMS. Both Rf genes, however, could accelate the development of abortive pollen in the case of WA-type CMS and their effect was additive. Crossing of CMS rice with chromosome segment substitution lines carrying Rf loci from IR24 resulted in similar pollen development and seed fertility.

Using a functional polymer containing nitrospirobenzopyran residues and poly(ethylene glycol), we developed a photoresponsive cell culture surface (PRCS), at which the cell adhesion can be enhanced locally by UV light irradiation (350–400 nm, 35 mW/cm2, 5 min) even on the region where living cells exist. Cell adhesion was evaluated by observing BALB/3T3 fibroblasts, which had been seeded on PRCS and then irradiated with UV light regionally, after 12 h incubation and subsequent washing to remove scarcely adhering cells. As a result, it was confirmed that the number of remaining cells in irradiated region was at most 2.5 times greater than that in nonirradiated regions, suggesting the implementation of a novel scheme to manipulate living cells individually by light irradiation in a parallel and simultaneous manner. Influence of the composition of the polymer material consisting the substrate surface was also investigated systematically. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 495–499, 2006

A 48-year-old woman was admitted to our emergency center for deterioration of consciousness. On arrival, her level of consciousness was 3 by the Japan Coma Scale and 10 by the Glasgow Coma Scale. Her blood pressure was 144/100 mmHg, and her pulse was regular at 136 beats/min. She was intubated under general anesthesia to lower the systolic blood pressure to 90-100 mmHg. Laboratory analysis showed elevated blood glucose (329 mg/dl), lactic dehydrogenase (311 U/l), creatinine kinase (CK, 410 U/l), and white blood cell count (19600/μl), and a reduced serum potassium (3.1 mmol/l). A computed tomographic scan showed a diffuse thick subarachnoid hemorrhage (SAH), and a chest X-ray revealed cardiomegaly and pulmonary edema. Electrocardiography on admission demonstrated sinus tachycardia and ST segment elevation in leads I, II, aVL, and V3 to V6, and a slightly prolonged QTc interval of 0.45 second. Echocardiography showed that wall motion of the left ventricular apex was significantly reduced, indicating specific “takotsubo” cardiomyopathy. Her cardiac function appeared to be well tolerant for direct surgery. Digital subtraction angiography was performed and disclosed an aneurysm at the P1 segment of the left posterior cerebral artery. Surgical intervention was undertaken by the left pterional approach. While a subarachnoid clot in the prepontine cistern was being evacuated to expose the aneurysm via an opticocarotid triangle, her blood pressure suddenly dropped to 20 mmHg, her heart rate declined to 20 beats/min, and carotid pulsation disappeared. The surgery was discontinued and cardiac resuscitation was conducted by intravenous injection of 1 mg epinephrine and cardiac massage by chest compression. Twenty-five minutes later her cardiac function stabilized, and the aneurysm was successfully clipped. The postoperative course was uneventful, and the electrocardiogram showed inversion of T waves and gradual improvement of echocardiographic abnormalities. An excessive catecholamine secretion related to hypothalamic damage is presumed to be the cause of cardiac dysfunction in the acute phase of SAH. Although evacuation of prepontine clot is a routine surgical maneuver, subtle compression or traction of the perforators from the P1 segment that feeds the hypothalamus might further injure the already affected hypothalamus to result in sudden cardiac arrest. In treatment of patients with SAH associated with cardiac dysfunction, preparation of an intra-aortic balloon pumping would be beneficial.

重症例に対しても待機せず可及的速やかに脳動脈瘤根治術を行うという方針のもと診断治療を行った,1996年1月から2000年12月までの5年間の235名のクモ膜下出血(SAH)患者につき検討した。患者の内訳は男性85名,女性150名,平均年齢60.8歳であった。来院時WFNS Grade (G)はGI 21名,GII 48名,GIII 2名,GIV 51名,GV 79名,および来院時心肺機能停止(CPA): 34名であり,GIV, GV, CPAが全体の70%を占めた。原則としてGIからGIVまでの全例,GVでは脳幹反射がみられるものを脳動脈瘤根治術の対象とし,待機せず直ちに診断,治療を開始した。CPAで早期に心肺蘇生が施行され自己心拍の再開をみたものはGVに準じた。167名(71.1%)に脳血管撮影(DSA), 146名(62.1%, 8名3.4%はDSA未施行)にクリッピング術,19名(8.1%)にGuglielmi detachable coil瘤内塞栓術を施行した。発症からの時間経過(中央値)は,来院まで47分,脳血管撮影まで200分,手術まで338分であった。1998年以降,21名に脳低温療法を行った。6か月後のGlasgow outcome scaleによる全体の転帰は,Good recovery (GR): 67名(29.5%), moderate disability (MD): 19名(8.4%), severe disability (SD): 38名(16.7%), persistent vegetative state (VS): 10名(4.4%), death (D): 93名(41.0%)であった。軽症例(GI, II, III)の転帰良好(GR+MD)は78.3%,不良(SD+VS) 10.1%,死亡11.6%,重症例(GIV, V)の転帰良好は24.8%,不良31.2%,死亡44%であった。CPAから蘇生後の4例に根治術施行,3名が生存,うち1名はGRであった。重症SAH症例が過半数を占める救命救急センターにおいて,“救いうる”SAH患者に一人でも多く良好な転帰を得るためには,来院後の再破裂予防と迅速な根治術施行とともに,低酸素脳障害の予防を含め,病院外救護体制の面からも取り組みが必要と思われた。

An antisense gene strategy was used to suppress the expression of rice allergen (RA) genes that comprise a multigene family. We produced transgenic rice plants harboring antisense genes that consisted of cDNA for a major RA driven by promoters of the RA, rice starch branching enzyme, rice prolamine and rice glutelin genes, which are specifically expressed in developing seeds. Among more than 100 transformants, the RA content in some transformants was reduced to no more than 20 % of that in non-transformant. However, an allergen-free transformant was not obtained. Since the transcribed antisense mRNAs are not fully used for suppression of the RA genes in transformants, we examined here the possibility of a spatial or temporal difference in the expression of the introduced antisense and endogenous RA genes. However, we found no remarkable difference in the place or timing of their expression. We also examined the effect of antisense genes on each of the genes in the RA family in transformants by twodimensional electrophoresis and immunoblotting. The results showed that the contents of the major RA and other RAs with nucleotide sequences that are highly homologous to the antisense sequence were markedly lowered, while the contents of other RAs with sequences showing relatively low homology to the major RA were hardly affected. These results suggest that the effect of the antisense gene on the RA gene family depends on the homology between the antisense sequence used and the target sequence.

Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products.

Soil salinization is a serious problem in agricultural lands worldwide. Understanding the mechanisms of salt-tolerant plants will contribute to knowledge necessary to genetically engineer salt-tolerant crops that grow on these saline lands. We identified a genotype of Sporobolus virginicus, a salt-tolerant turf grass, that showed a salinity tolerance to up to a three-fold higher NaCl concentration than seawater salinity. In addition to salt secretion from salt glands on the leaves, this genotype accumulated K+ and proline, a compatible solute, to higher levels than other genotypes under salinity. These properties must contribute to the advanced salt tolerance of this genotype., Understanding the mechanisms used by halophytic members of the Poaceae to cope with salt stress will contribute to the knowledge necessary to genetically engineer salt-tolerant crops. In this study, we identified a genotype of Sporobolus virginicus, a halophytic turf grass collected in Japan, and investigated its growth rate, ion concentration and secretion, and proline concentration in comparison with the reported properties of genotypes collected from the USA, South Africa and Egypt. Surprisingly, the Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a 3-fold higher concentration than seawater salinity. Shoot growth was stimulated by 100 mM NaCl and root growth was stimulated at salinities of up to 1 M NaCl. Accumulation of Na+ and CI− in shoots and roots was rapidly elevated by salinity stress but did not exceed the levels required for osmotic adjustment, due in part to ion secretion by salt glands, which are present in genotypes of S. virginicus. However, the Japanese genotypes accumulated K+ to a higher level than other genotypes, resulting in a relatively high K+/Na+ ratio even under salinity stress. An increase in proline concentration was observed that was proportional to the NaCl concentration in the culture solution and might partially account for osmotic adjustment in the shoots. We also generated and characterized cultured cells of S. virginicus. In 500 mM NaCl, the cultured cells showed an enhanced growth compared with cultured cells of rice. The concentration of Na+ and CI− in the cultured cells in 300–500 mM NaCl was lower than in 100 mM NaCl. Cultured cells of S. virginicus accumulated proline to higher levels than rice cells cultured under salinity stress. The active regulation of Na+, Cl− and K+ influx/efflux and proline accumulation might be involved in salt tolerance mechanisms at the cellular level as well as in planta.

Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PHD) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PHD gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.

We retrospectively analyzed and compared overall outcome of poor-grade patients (WFNS Grade IV, V, and cardiopulmonary arrest on arrival |CPAOA|) with subarachnoid hemorrhage (SAH) in 2 study periods. The first was from July 1993 to June 1994 and the second from July 1998 to June 1999. A total of 20 patients, 7 with Grade IV, 5 with Grade V, and 8 with CPAOA were included in the first period; and a total of 36 patients, 6 with Grade IV, 21 with Grade V, and 9 with CPAOA, in the second period. The major recent changes in the treatment protocol were 1) deep sedation, intensive control of blood pressure, and endotracheal intubation in the emergency room in the resuscitative phase; 2) immediate cerebral angiography under general anesthesia after diagnosis of SAH; 3) aneurysm obliteration by Guglielmi detachable coil (GDC) as an option; and 4) early enteric feeding and tracheostomy for patients in a prolonged comatose state. Therapeutic mild brain hypothermia was conducted in 3 Grade V patients in the second-period study. Aneurysm was obliterated by clipping in 7 and 18 patients in each period and by GDC in 4 patients in the second period. The median time from onset to admission, to angiography, and to surgery was 29, 440, and 797.5 minutes, respectively, in the first period, and 32, 99, and 210 minutes, respectively, in the second period. Overall outcome assessed at 3 months by the Glasgow Outcome Scale was; GR: 2 (10%), MD: 0 (0%), SD: 1 (5%), VS: 2 (10%), D: 15 (75%) for the first period and GR: 5 (13.9%), MD: 3 (8.3%), SD: 7 (19.4%), VS: 1 (2.8%), D: 20 (55.6%) for the second period. A favorable outcome (GR+MD) increased from 10% to 22.2%, and deaths were reduced from 75% to 55.6% (P=0.02, chi-square test). Angiography and surgical intervention immediately after resuscitation produce a more favorable outcome for severe SAH patients without increasing the risk of rerupture.

A 77-year-old woman suddenly lost consciousness and presented with right hemiparesis. Computed tomography showed a large subcortical hematoma in her left frontal lobe associated with subarachnoid hemorrhage. The first impression was hemorrhage due to a ruptured aneurysm of the middle cerebral or the internal carotid artery on the left. Left internal carotid angiography showed no aneurysm or vascular anomaly, but back flow of contrast medium into the external carotid artery disclosed two saccular aneurysms arising from the anterior branch of the left middle meningeal artery (MMA). Emergent surgical intervention confirmed that the hematoma was due to ruptured MMA aneurysm. Nontraumatic MMA aneurysm should be recognized as a possible causative lesion of intracranial hemorrhage despite the extremely low incidence. External carotid angiography should be performed in patients with intracranial hemorrhage in whom no vascular cause can be detected in the internal carotid system.

最重症くも膜下出血(SAH)に対する可及的早期脳低温療法の有用性の検討するため,1999年4月以降に経験した75歳未満のWFNS Grade VのSAH 16名に診断確定直後より脳低温療法を施行し,脳動脈瘤根治術を施行した。内訳は男性10名,女性6名,年齢50-72歳(平均63.1歳),Glasgow Coma Scale (GCS)は,心肺停止から蘇生しGCS 3となったもの2名,3が6名,4が1名,5が3名,6が4名であった。CT上4名が10mm以上の正中偏位を伴う血腫を合併し,4名が鋳型状脳室内血腫を伴った。SAHの診断確定後,速やかに脳低温療法を開始し,クリッピング術,外減圧術を施行した。経過時間(中央値)は発症後搬入まで33分,脳血管撮影まで86分(7名は3D-CTAのみ施行),手術まで174分であった。33-34℃の脳低温療法を2-3日施行後,復温した。復温終了まで平均8.3日であった。発症24時間以内に経腸栄養を開始,穿刺式気管切開を行い肺理学療法を徹底した。脳低温療法施行16名のGlasgow Outcome Scaleによる転帰はgood recovery (GR) 2名,moderate disability (MD) 3名,severe disability (SD) 8名,persistent vegetative state (VS) 2名,death (D) 1名であった。脳低温療法の合併症による症状の悪化や中断例はなく,一次性脳損傷が転帰不良の原因と考えられた。脳低温療法導入直前のGrade VのSAH連続24名と導入後,脳低温の適応外となった6名を含む連続22名を比較すると,転移良好(GR+MD)率に関しては有意差は認めなかったが(12.5% vs. 22.7%), VS+Dの率は71.8%から29.2%と有意に低下した。最重症SAHに対する超急性期からの脳低温療法は少なくとも植物症への移行と死亡を減少させ,一次性脳損傷を軽減させる可能性がある。

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.

An antisense gene strategy was applied to suppress the 14–16 kDa allergen gene expression in maturing rice seeds. Gene constructs producing antisense RNAs of the 16 kDa allergen under the control of some rice seed-specific promoters were introduced into rice by electroporation. Immunoblot and RNA blot analyses of the seeds from the transgenic rice plants using the allergen-specific monoclonal antibody and a sequence-specific antisense RNA probe demonstrated that the 14–16 kDa allergen proteins and their transcripts of the seeds from several transgenic lines were present in much lower in amounts than those of the seeds from parental wild-type rice. The high levels of reduction observed were stably inherited in at least three generations.

A translational initiation sequence, conserved among soybean lipoxygenase genes, was fused with the β-glucuronidase (GUS) gene, placed under 35S cauliflower mosaic virus promoter control, then electroporated into suspension-cultured protoplasts of tobacco and rice. These constructs produced 11- and 7-fold increases in GUS activity, respectively, compared with the original GUS construct. The transcriptional activity of the modified GUS gene fused with the TATA box from a soybean lipoxygenase gene was also detected with great sensitivity. The fusion construct is useful for detectingcis-regulatory element effects on transcription, in particular, silencers.

The beta-glucuronidase (GUS) gene linked to the promoter of rice light harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) was introduced into rice by electroporation. Expression of the GUS gene with the LHCP promoter in transformed protoplasts and callus was much lower than with the cauliflower mosaic virus (CaMV) 35S promoter. In green organs, however, LHCP-GUS activity with the LHCP promoter was 10-fold higher than that with the CaMV 35S promoter. Expression of the LHCP-GUS gene was detected in leaves, stems and floral organs but not in roots. Histochemical analysis of leaves showed that the GUS gene was expressed specifically in green tissues. In selfed seeds of the transformants, GUS gene expression was detectable only in the germinated embryos after greening. Northern blot analysis revealed that transcription of the GUS gene with the LHCP promoter was not detectable in dark-grown seedlings of transformants but was induced by exposure to light. These results show that regulation of the introduced chimeric gene in transgenic plants was characteristic of the LHCP gene itself.

Polymerase chain reaction (PCR) is an efficient method for obtaining a desired nt sequence if both required primers can hybridize to the target DNA molecule specifically. A rapid and simple PCR-based method for analyzing plasmids using intact cells was established. An attempt to target a rice waxy sequence by PCR using homologous primers was also carried out. In three cases, specific fragments were amplified and their nucleotide sequences were determined. However, the cloned rice waxy gene contained base substitution mutations. The cumulative frequency of mutation after 30 polymerization cycles was estimated to be one in 500 bp.

We have developed a method for reproducibly obtaining transgenic rice at a high frequency (10(-4)): electroporation with a buffer in which chloride ions are replaced with organic acids. Co-transformation frequencies of the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes located on two separate plasmids were higher than 50%. Transgenic rice plants contained multiple copies of introduced genes integrated into their genomes in a complex manner. GUS enzyme activity was not proportional to gene copy number. Introduced HPT genes were detected and expressed in the progeny of transformants.

A simulation study was undertaken using a compartmental flow model of a large high-flow cerebral arteriovenous malformation to investigate the hemodynamic changes during obliteration procedures. Under certain autoregulatory conditions, marked hyperperfusion (92 ml/100 g/min) could be induced in association with increased wall stress of the arterioles. Narrowing of the autoregulatory pressure range and its shift to a low pressure level are suspected to be among the possible causes of normal perfusion pressure breakthrough phenomenon.