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Pseudomonas cannabina pv. alisalensis (Pcal), which causes bacterial blight of brassicaceous plants, is an economically important pathogen worldwide. Copper fungicide and antibiotics are major strategies to manage the disease caused by Pcal; however, a Pcal strain resistant to these chemicals has already been found, and severe outbreaks of bacterial blight have been reported on cabbage in Japan. Therefore, there is an urgent need to develop new Pcal management strategies. Plant defense activators could be useful not only to protect plants against invading pathogens, but also to reduce the amount of copper fungicides and antibiotics applied. However, the mechanisms by which plant defense activators contribute to controlling diseases remains unclear. In this work, we focused on cabbage and acibenzolar-S-methyl (ASM), a well-known plant defense activator. Expression profiles revealed that ASM induced expression of systemic acquired resistance (SAR) marker genes including PR1, PR2, and PR5 in cabbage plants. We also demonstrated that a soil drench with ASM 2 h before transplanting clearly reduced bacterial blight symptoms and reduced Pcal bacterial populations in cabbage. ASM application was also able to prime cabbage for Pcal resistance by activating stomatal-based defense. Our findings highlight that ASM protects plants from bacterial pathogens by activating stomatal-based defense.

The wind characteristics of downbursts are quite different from those of turbulent boundary layers. Non-stationarity (e.g., a severe moving downdraft and a strong divergent outflow near the ground) causes severe damage to buildings and structures. In our previous study, the following features of non-stationarity were experimentally reproduced using a downburst simulator: wind speed increase that occurs when the wind hits the ground, wind speed increase resulting from the movement of a downdraft accompanied by that of parent clouds, and wind speed increase caused by a combination of these phenomena. In the experiments, the measurement area of the flow fields and the points of pressure measurements on a building model were so limited that detailed information on the flow and pressure fields could not be obtained. Therefore, in the present, we conduct a computational fluid dynamics (CFD) simulation of downbursts using a large eddy simulation to investigate the mechanism that causes strong winds. We focus on the effects of the aforementioned non-stationarity of downbursts on the wind loads on buildings. The CFD simulation successfully reproduces the experimental results and clarifies the features of downburst-induced wind loads.

In many non-cyclone areas around the world, severe thunderstorm downbursts can produce annual gust peaks that are the design wind speeds of general structures, and the resultant wind loads pose a major potential threat to critical infrastructure, such as transmission line structural system. The flow fields generated by downbursts are different from those by the synoptic winds in the atmospheric boundary layer. In order to obtain a wind load model of downburst, scholars have made many attempts to study the flow characteristics of downburst-like events. However, in previous studies, the effects of different experimental setup parameters on distribution of downburst wind speed have not been fully discussed. In this paper, an experimental study is conducted using a pulsed wall jet experimental simulator located at Tohoku University, Japan, to analyse the flow fields of stationary and travelling downbursts, and the resultant wind load effects on transmission line structural system. The comparison results show that different experimental settings will affect the maximum wind speed obtained and the height providing the maximum wind speed. The translational speed ratio defined by the ratio of moving speed to jet inlet speed will affect the wind speed distribution in the longitudinal direction of the generated flow field of the travelling downburst. In addition, the comparative studies show that the wind speed distribution of travelling downburst cannot be simply expressed as the vector sum of stationary downburst wind speed distribution and the jet moving speed, and that speed of the travelling downburst can intensify the non-uniform wind loading relaying to the transmission line structural system. For the analysis of the downburst flow fields, a multi-degree-of-freedom analysis is needed to consider its three-dimensional effect, that is, the distributions of wind speeds along the three coordinate axes, and the comprehensive effects of influencing parameters. Refereed/Peer-reviewed

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

Background The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. Methods Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. Results PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-β-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-β-induced EMT induction. Conclusions These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.

Metabolic reprogramming contributes to dynamic alteration of cell functions and characteristics. In T cells, TCR-mediated signaling evokes metabolic reprogramming and autophagy. AMBRA1 is known to serve in the facilitation of autophagy and quality control of mitochondria, but the role of AMBRA1 in T cell metabolic alteration is unknown. Here, we show that AMBRA1, but not ATG7, plays a role in TCR-mediated control of glycolytic factors and mitochondrial mass, while both AMBRA1 and ATG7 are required for autolysosome formation. Our results suggested that AMBRA1 is a core factor that controls both autophagy and metabolic regulation.

Metabolic syndrome (MS), overlapping type 2 diabetes, hyperlipidemia, and/or hypertension, based on high-fat diet, poses risk for cardiovascular disease. A critical feature associated with such risk is the functional impairment of endothelial progenitor cells (EPCs). Dipeptidyl dipeptidase-4 inhibitors are known not only to inhibit degradation of incretins to control blood glucose levels, but also to improve EPC bioactivity and induce anti-inflammatory effects in tissues. In the present study, we investigated the effects of such an inhibitor, MK-06266, in ischemia model of MS using diet-induced obese (DIO) mice. EPC bioactivity was examined in MK-0626-administered DIO mice and non-treated control group, using an EPC colony-forming assay and bone marrow cKit+ Sca-1+ lineage-cells, and peripheral blood-mononuclear cells. Our results showed that, in vitro, the effect of MK-0626 treatment on EPC bioactivities and differentiation was superior in comparison with non-treatment. Further, in vivo hindlimb ischemia model experiment indicated that microvascular density and pericyte-recruited arteriole number were increased in MK-0626-administered group, but not control group. Lineage profiling of isolated cells from ischemic tissues disclosed that MK-0626 administration has an inhibitory effect on unproductive inflammation. This occurred via a decrease in the influx of total blood cells and pro-inflammatory cells such as neutrophils, total macrophages, M1, total T-cells, cytotoxic T-cells, and B-cells, with a concomitant increase in number of regeneration-associated cells, such as M2/M ratio and Treg/T-helper. Laser Doppler analysis revealed that at day 14 after ischemic injury, blood perfusion in hindlimb was grater in DIO mice treated with MK-0626, but not in control. In conclusion, the dipeptidyl dipeptidase-4 inhibitor has a positive effect on EPC differentiation in MS model of DIO mice. Following ischemic injury, DPP-4 i sharply reduces recruitment of pro-inflammatory cells into ischemic tissue, and triggers regeneration and reparation process. Thus, DPP-4 i is a promising therapeutic agent for MS treatment.

The objective of this study was to determine the effects of geometry on the wind loads acting on photovoltaic panel arrays with modules mounted parallel to roof surfaces of low-rise buildings. Specific attention was made to determine the effects of varying the spacing between individual modules, G, and the mounting height above the roof surface, H. The photovoltaic system was modeled as an array of 28 modules on a 1/20 scale building model with a roof slope of 30°. In addition, limited studies were carried out with the array mounted on a flat-roof to assess the impact of roof slope. In general, it was found that larger gaps between modules, G, and smaller gaps between the panels and the roof surface, H, were found to yield lower net wind loads. Minimum loads tend to occur for G/H>~1, for the particular panel size considered in the study. Pressure equalization between the upper and lower surfaces of the modules results in the magnitudes of the net panel pressures typically being lower than those for the bare roof surface. A pressure equalization factor, Ceq, was used as a measure of how much the peak net wind loads on the panels are reduced relative to the peak external loads.

To investigate the immunological changes caused by severe sepsis in elderly patients.One-year, prospective observational study.Emergency department and intensive care unit of a single university hospital.Seventy-three patients with severe sepsis and 72 healthy donors.In elderly septic patients (aged 65 yr and over), 3-month survival was significantly reduced compared with that for adult patients (18-64 yr) (60% vs. 89%, p0.01). We found that lymphopenia was prolonged for at least 21 days in elderly nonsurvivors of sepsis, while the number of lymphocytes recovered in both adult and elderly survivors of sepsis. In order to examine the immunological status of septic patients, blood samples were collected within 48 hrs of diagnosis of severe sepsis, and peripheral blood mononuclear cells were purified for flow cytometric analysis. T cell levels were significantly reduced in both adult and elderly septic patients, compared with those in healthy donors (56% and 57% reduction, respectively). Interestingly, the immunocompetent CD28+ subset of CD4+ T cells decreased, whereas the immunosuppressive PD-1+ T cells and the percentage of regulatory T cells (CD4+ T cells that are both Foxp3+ and CD25+) increased in elderly patients, especially nonsurvivors, presumably reflecting the initial signs of immunosuppression.Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G1 cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G1-phase extension and represses CD8 expression in a G1 extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G1 extension and CD8 repression. Importantly, forced G1 extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G1 progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G1-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity. Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of γ-tubulin-independent microtubules in early embryos; however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the γ-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.

Small cell (endocrine cell) carcinoma of the gallbladder in a 62-year-old woman is reported. The palliative cholecystectomy specimen revealed a submucosally invading tumor with extensive hemorrhagic necrosis. At autopsy, performed five months after surgery, a huge tumor measuring 14 x 12 x 8 cm was located at the liver hilus. No signs or symptoms related to overproduction of hormones were recorded throughout her illness. Neither lung lesions nor gall stones were identified. Histologically, diffuse proliferation of small, spindle-shaped atypical tumor cells with numerous mitoses was evident. Intraepithelial tumor cell proliferation in the gallbladder mucosa was seen focally. The neuroendocrine nature of the tumor cells was confirmed by the histologic pattern of growth with pseudo-rosette formation, positive reaction for Grimelius' argyrophilia, neuron-specific enolase and Leu 7, and ultrastructural demonstration of neuroendocrine-type granules. Immunostaining for a variety of hormones was all negative. Characteristically, foci with squamous and adenocarcinomatous differentiation were identified in the tumor tissue. The glandular components were immunoreactive for carcinoembryonic antigen, secretory component, epithelial membrane antigen and CA19-9. The histogenesis and totipotentiality of the neoplasm were discussed.

Aims: Frequent onset of several mental disorders starts around undergraduate age for university students. Mental health check-ups of the new students might help provide them with useful supports for improving their mental health. However, few studies have examined the validity of the check-up methods. Methods: Whether the scores of a five-factor personality inventory (NEO-FFI) at matriculation predict the needs of mental care and treatment during the first year of the undergraduate course were examined in 8287 new students of a university in Tokyo. Results: Logistic regression showed that high neuroticism, low extraversion and high openness of NEO-FFI, majoring in literature/philosophy/ psychology and living out of home were associated with need for mental care/treatment, in addition to the previous use of mental care services. Conclusions: Personality inventory such as five-factor ones may be a useful supplemental tool for mental health check-up at matriculation to predict future needs of mental support in undergraduate university students. Students who smoke, live alone out of home and major in subjects such as philosophy might need to be more carefully supported than other students.

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.

The relative-rate method has been used to determine the rate coefficients for the reactions of OH radicals with three C5 biogenic alcohols, 2-methyl-3-buten-2-ol (k1), 3-methyl-3-buten-1-ol (k2), and 3-methyl-2-buten-1-ol (k3), in the gas phase. OH radicals were produced by the photolysis of CH3ONO in the presence of NO. Di-n-butyl ether and propene were used as the reference compounds. The absolute rate coefficients obtained with the two reference compounds agreed well with each other. The O3 and O-atom reactions with the target alcohols were confirmed to have a negligible contribution to their total losses by using two kinds of light sources with different relative rates of CH3ONO and NO2 photolysis. The absolute rate coefficients were obtained as the weighted mean values for the two reference compound systems and were k1 = (6.6 ± 0.5) × 10−11, k2 = (9.7 ± 0.7) × 10−11, and k3 = (1.5 ± 0.1) × 10−10 cm3 molecule−1 s−1 at 298 ± 2 K and 760 torr of air. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 379–385 2004

Background Cell‐based therapies involving mononuclear cells ( MNC s) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNC s has not been evaluated. We investigated the therapeutic potential of peripheral blood ( PB ) MNC s, operated by recently developed quality and quantity ( QQ ) culture of endothelial progenitor cells ( EPC s). Methods and Results PBs were collected from healthy volunteers; peripheral blood mononuclear cells ( PBMNC s) isolated from these PB s were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt‐3 ligand, vascular endothelial growth factor, and interleukin‐6. The resulting cells ( QQMNC s) in EPC colony‐forming assay generated significantly more definitive EPC colonies than PBMNC s. In flow cytometry, macrophages and helper T lymphocytes of QQMNC s became phenotypically polarized into angiogenic, anti‐inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T‐cell expansion. Quantitative real‐time polymerase chain reaction ( qRT ‐ PCR ) assay revealed the predominant proangiogenic gene expressions in QQMNC s versus PBMNC s. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx , cultured “early EPC ” Tx ( eEPCTx ), and granulocyte colony‐stimulating factor mobilized CD34 + cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCT x, but also earlier than after GmCD34Tx. Histological evaluations and q RT ‐ PCR assays in ischemic hindlimbs demonstrated that QQMNCTx , similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx . Conclusions QQ culture potentiates the ability of PBMNC s to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture‐treated PBMNC s may provide a promising therapeutic option for ischemic diseases. Clinical Trial Registration URL : irb.med.u-tokai.ac.jp/d/2/monthly/2010.html ; IRB No.: 10R‐020. URL : irb.med.u-tokai.ac.jp/d/2/monthly/201312.html ; IRB No.: 13R228.

Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.

Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.

Using the relative-rate technique, the rate constant for the reaction of OH radicals with isoprene was determined in one atmosphere of air at 298±2 K. Methyl nitrite photolysis in air was used as a source of OH radicals. Ozone and O-atom reactions with isoprene were confirmed to have a negligible contribution on the loss of isoprene. The rate constant relative to three reference compounds yielded (10.4±0.4)×10−11 cm3 molecule−1 s−1 (the uncertainty is 2σ).