Don't move during ablation of atrial fibrillation!

BACKGROUND: Restless patient is recalcitrant during ablation of atrial fibrillation (AF). We aimed to assess the association between patient movements during AF ablation and its outcome. METHODS: We examined the body movement during AF ablation in 78 patients with the use of a novel portable respiratory monitor, the SD-101, which also has the ability to quantify the frequency of body movements. RESULTS: The body movement index, defined as the number of the units of time with body movement events divided by the recording time (11.4 ± 6.5 events/h), was significantly correlated with the ablation time defined as the time from the first point of the ablation to the end of the procedure (1.2 ± 0.3h) (r=0.35; p=0.0014) and a total radiofrequency energy applied (56.6 ± 17.7 kW) (r=0.36; p=0.0015). A multiple linear regression analysis showed that non-paroxysmal AF (ß=0.25; p=0.036) and the body movement index (ß=0.36; p=0.0019) were independent determinants of the ablation time. The body movement index was similar in patients with and without recurrence of AF. CONCLUSIONS: Keeping patients motionless may be important to reduce the procedural duration of AF ablation.

Clinical significance of sleep-disordered breathing induced by sedatives during ablation of atrial fibrillation.

BACKGROUND: Sleep-disordered breathing (SDB) frequently is induced by sedation during ablation of atrial fibrillation (AF). We tested whether or not sedative-induced SDB is associated with clinical sleep apnea syndrome (SAS). METHODS: We examined SDB observed during intra-ablation sedation with a novel portable respiratory monitor (PM), SD-101, in 140 patients undergoing AF ablation without any known SAS. A sleep study was repeated the next night of the ablation with the simultaneous use of the SD-101 and type 3 PM, APNOMONITOR V. RESULTS: The respiratory disturbance index (RDI) during sedation (20.0±7.8 events/h) was significantly correlated with the RDI measured by the SD-101 during the night (15.8±13.7 events/h; r=0.50) and the RDI assessed by the APNOMONITOR V during the night (12.7±12.3 events/h; r=0.55). An excellent agreement was found between the RDIs simultaneously measured with the 2 PMs (intraclass correlation coefficient, 0.84), especially for an RDI of ≥30 events/h (κ statistic value, 0.82). The area under the receiver-operating characteristic curve for the RDI with the use of the SD-101 during sedation to identify the patients with an RDI of ≥30 events/h by both PMs during the night was 0.92. A left atrial diameter of >40 mm (odds ratio [OR], 4.10) and an RDI during sedation of >20 events/h (OR, 17.75) were independently associated with having an RDI of ≥30 events/h with both PMs during the night. CONCLUSIONS: Frequent episodes of sedative-induced SDB may have a diagnostic value for SAS in patients with AF.

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS.

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells.

Manual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semi-automatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.

An unidentified ultraviolet-B-specific photoreceptor mediates transcriptional activation of the cyclobutane pyrimidine dimer photolyase gene in plants.

Cyclobutane pyrimidine dimers (CPDs) constitute a majority of DNA lesions caused by ultraviolet-B (UVB). CPD photolyase, which rapidly repairs CPDs, is essential for plant survival under sunlight containing UVB. Our earlier results that the transcription of the cucumber CPD photolyase gene (CsPHR) was activated by light have prompted us to propose that this light-driven transcriptional activation would allow plants to meet the need of the photolyase activity upon challenges of UVB from sunlight. However, molecular mechanisms underlying the light-dependent transcriptional activation of CsPHR were unknown. In order to understand spectroscopic aspects of the plant response, we investigated the wavelength-dependence (action spectra) of the light-dependent transcriptional activation of CsPHR. In both cucumber seedlings and transgenic Arabidopsis seedlings expressing reporter genes under the control of the CsPHR promoter, the action spectra exhibited the most predominant peak in the long-wavelength UVB waveband (around 310 nm). In addition, a 95-bp cis-acting region in the CsPHR promoter was identified to be essential for the UVB-driven transcriptional activation of CsPHR. Thus, we concluded that the photoperception of long-wavelength UVB by UVB photoreceptor(s) led to the induction of the CsPHR transcription via a conserved cis-acting element.

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction.

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.

USF is involved in the transcriptional regulation of the chipmunk HP-25 gene.

The hibernation-specific HP-25 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. Our previous studies have revealed two positively acting transcriptional regulatory regions in the 5'-flanking region of the chipmunk HP-25 gene, one from -260 to -80 and another from -80 to -59, and a pivotal role for hepatocyte nuclear factor-4 (HNF-4), which binds to the proximal regulatory region, in HP-25's liver-specific transcription. A database search for transcription factor binding sites in the distal regulatory region indicated the presence of two potential binding sites for upstream stimulatory factor (USF): one between -161 and -156 and the other between -143 and -138. In an electrophoretic mobility shift assay (EMSA), in vitro-translated USF bound only to the sequence from -143 to -138. USF did not bind the corresponding sequence of the tree squirrel HP-25 gene, which has two base substitutions. Transient transfection studies in COS-7 cells showed that USF could activate the transcription of the chipmunk HP-25 gene, and that tree squirrel-type base substitutions in the USF-binding site aborted the transactivation by USF. By chromatin immunoprecipitation (ChIP) analysis, we confirmed that USF bound to the promoter region of the HP-25 gene in the chipmunk liver, and not in the kidney or heart. These results indicate that USF is involved in the transcriptional regulation of the chipmunk HP-25 gene in the liver, and that the base substitutions in the USF-binding site contribute to the lack of HP-25 gene expression in the tree squirrel.

Induction and inhibition of cyclobutane pyrimidine dimer photolyase in etiolated cucumber (Cucumis sativus) cotyledons after ultraviolet irradiation depends on wavelength.

Under polychromatic ultraviolet (UV) irradiation (maximum energy at 327 nm) the activity of DNA photolyase specific to cyclobutane pyrimidine dimers (CPDs), CPD photolyase, increased by an amount which depended on UV irradiance, and the level of CPD photolyase gene (CsPHR) transcripts temporarily increased before the activity reached a constant level. UV light (>320 nm) was more effective than visible light at increasing CPD photolyase activity. In contrast, monochromatic UV irradiation at wavelengths <300 nm increased the level of CsPHR transcripts similarly to irradiation at wavelengths >320 nm, but reduced CPD photolyase activity compared with the dark control. Exposure of a CPD photolyase solution to UV-C (254 nm) reduced enzyme activity and induced accumulation of H(2)O(2). Addition of H(2)O(2) to the enzyme solution also inactivated CPD photolyase activity. These results suggest the possibility that reactive oxygen species participate in the inactivation of CPD photolyase in cotyledons exposed to UV irradiation of <300 nm.

Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis.

Cytokinins and auxins are major phytohormones involved in various aspects of plant growth and development. These phytohormones are also known to antagonize the effects of abscisic acid (ABA) on stomatal movement, and to affect ethylene biosynthesis. As ethylene has an antagonistic effect on ABA-induced stomatal closure, the possibility that the antagonistic effects of these phytohormones on ABA were mediated through ethylene biosynthesis was investigated. Both the cytokinin, 6-benzyladenine (BA), and the auxin, 1-naphthaleneacetic acid (NAA), antagonized ABA-induced stomatal closure in a manner similar to that following application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). However, these effects were negated when ethylene signalling, perception, or biosynthesis were blocked. As stomatal aperture is regulated by changes in guard cell volume, ABA application was found to reduce the volume of the guard cell protoplasts (GCP). It was found that BA, NAA, or ACC application compensated perfectly for the reduction in GCP volume by ABA application in WT plants. The above observations suggest that cytokinins and auxins inhibit ABA-induced stomatal closure through the modulation of ethylene biosynthesis, and that ethylene inhibits the ABA-induced reduction of osmotic pressure in the guard cells.

Circannual control of hibernation by HP complex in the brain.

Seasonal hibernation in mammals is under a unique adaptation system that protects organisms from various harmful events, such as lowering of body temperature (Tb), during hibernation. However, the precise factors controlling hibernation remain unknown. We have previously demonstrated a decrease in hibernation-specific protein (HP) complex in the blood of chipmunks during hibernation. Here, HP is identified as a candidate hormone for hibernation. In chipmunks kept in constant cold and darkness, HP is regulated by an individual free-running circannual rhythm that correlates with hibernation. The level of HP complex in the brain increases coincident with the onset of hibernation. Such HP regulation proceeds independently of Tb changes in constant warmth, and Tb decreases only when brain HP is increased in the cold. Blocking brain HP activity using an antibody decreases the duration of hibernation. We suggest that HP, a target of endogenously generated circannual rhythm, carries hormonal signals essential for hibernation to the brain.

Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis.

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethylene-overproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.

[Investigation of mechanisms of mammalian hibernation and its possible application in medical treatment].

Mammalian hibernation has been reported to increase resistance to various harmful events such as low body temperature, severe ischemia, bacterial infection, irradiation, and muscle disuse, and to prolong the lifespan of the mammal. Therefore, hibernation mechanisms are thought to play a critical role in maintaining healthy organisms. Although the application of this physiological phenomenon to medical fields has strongly been desired, it has been prevented by a poor understanding of the hibernation mechanism. In order to clarify how mammalian hibernation is controlled in organisms, we have looked for a physiological signal of hibernation and found marked changes in cardiac calcium regulation associated with a circannual hibernation. Focusing on these changes, we initially discovered a molecular marker of hibernation, hibernation-specific proteins (HP), of which production in the liver and the blood content are controlled by an endogenous circannual rhythm responsible for hibernation. Our recent studies on HP regulation have revealed that circannual signals for the timing of hibernation are transmitted through the neuroendocrine system and that HP are actively transported into the cerebrospinal fluid (CSF) prior to the onset of hibernation. This suggested that hibernation is controlled by HP in the brain and its regulation system. Based on these results, the future medical application of these results is discussed.

HNF-1 regulates the promoter activity of the HP-27 gene.

The hibernation-specific HP-27 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. In the promoter region, the chipmunk gene has a potential HNF-1 binding site, and the tree squirrel gene has two base substitutions in the corresponding sequence. In this paper, we investigated the role of HNF-1 in the HP-27 gene promoter activity. Gel retardation assays with in vitro-translated HNF-1 and super-shift assays using HepG2 nuclear extracts and an anti-HNF-1 antibody revealed that HNF-1 bound to the chipmunk gene sequence. HNF-1 also bound to the tree squirrel sequence, but with much lower affinity. In HepG2 cells, HNF-1 activated transcription from the chipmunk HP-27 gene, but not from the tree squirrel gene. In addition, the tree squirrel-type base substitutions in the HNF-1 binding site greatly reduced the promoter activity of the chipmunk HP-27 gene. These results indicate that HNF-1 is required for the promoter activity of the chipmunk HP-27 gene, and that the base substitutions in the HNF-1 binding site are involved in the lack of HP-27 gene expression in the tree squirrel.

Analysis of gene structures and promoter activities of the chipmunk alpha(1)-antitrypsin-like genes.

The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.

A physiological and morphological study on the injury caused by exposure to the air pollutant, peroxyacetyl nitrate (PAN), based on the quantitative assessment of the injury.

A method for the numerical assessment of the foliar injury caused by the photochemical oxidant, peroxyacetyl nitrate (PAN), was devised, using three injury indices: fresh weight (FW) loss, decreased photosynthetic pigment content, and increased ion leakage, which can be measured using the same leaves. The injury indices clearly indicated a larger number of PAN-sensitive leaves and a more severe level of injury in the PAN-sensitive variety of Petunia hybrida, White Champion (WHITE), compared to the PAN-tolerant variety, Blue Champion (BLUE). FW and photosynthetic pigment content decreased correlatively in both varieties, but ion leakage increased only in WHITE. Morphological observations revealed that ion leakage started concurrently with the start of plasmolysis-like symptoms at the mesophyll cells of injured WHITE leaves, whereas FW loss corresponded to the shrinkage of cells without loss of their round shape in BLUE leaves. PAN injuries measured by the injury indices were markedly increased in the presence of light, and the morphological changes following PAN exposure were similar to those caused by the superoxide-generating chemical, paraquat. The results suggested that PAN injuries indicated by the three injury indices are all light-dependent, but are caused through several independent mechanisms.

Genomic structure of the cucumber CPD photolyase gene.

Light-dependent transcriptional activation of the photolyase gene imparts UVB tolerance to a plant. In the present study, the cucumber CPD photolyase gene (CsPHR) was isolated from a genomic DNA library and its genomic structure was scrutinized. As a result, putative light-responsive cis-acting elements were found in the CsPHR promoter.