Acute exercise increases expression of extracellular superoxide dismutase in skeletal muscle and the aorta

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise.     

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

Gamma-secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.     

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson’s disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.     

Efficient monitoring of maize orange leafhopper, <Emphasis Type="Italic">Cicadulina bipunctata</Emphasis> (Hemiptera: Cicadellidae), and small brown planthopper, <Emphasis Type="Italic">Laodelphax striatellus</Emphasis> (Hemiptera: Delphacidae), in forage maize fields using yellow sticky traps

The monitoring of insect pests in fields of forage maize is difficult because plants are tall and grow at a high density. We investigated the effectiveness of colored sticky traps and appropriate conditions for monitoring insect pests in forage maize fields. Large numbers of the maize orange leafhopper, Cicadulina bipunctata Melichar (Hemiptera: Cicadellidae), and the small brown planthopper, Laodelphax striatellus Fallen (Hemiptera: Delphacidae), were collected during the experimental period with yellow and blue sticky traps placed in summer crop forage maize fields. A greater number of insects were trapped in yellow traps relative to blue traps. Traps located at a lower height (40 cm above the ground) attracted larger numbers of C. bipunctata, whereas L. striatellus did not demonstrate a height-dependent preference. These results indicated that yellow-colored sticky traps located at low height are effective for collecting C. bipunctata and L. striatellus simultaneously. Seasonal occurrence data obtained by the yellow sticky traps showed clearer seasonal occurrences than that obtained by two previously developed methods, suction and light traps, indicating that sticky traps are effective for monitoring the seasonal occurrence of these two insects in forage maize fields.     

Reproduction,grazing, and development of the large subarctic calanoid <Emphasis Type="Italic">Eucalanus bungii</Emphasis>: is the spring diatom bloom the key to controlling their recruitment?

The response of large calanoid, Eucalanus bungii, to environmental fluctuation, particularly in relation to the spring diatom bloom in the Oyashio region, western subarctic Pacific Ocean, was examined by investigating egg production, grazing, development and starvation tolerance. Mean in situ egg production rate increased with ambient chlorophyll-a concentration, ranging from 0 to 47 eggs female⁻¹ d⁻¹, while no diurnal synchronous spawning behavior was observed. Under the spring bloom condition, E. bungii showed prey preference for less mobile and larger-sized prey (≥30 μm ESD) and bloom-forming diatom Thalassiosira spp. accounted for >80% of ingested carbon. In the laboratory, E. bungii was successfully reared from newly hatched nauplii to adult with the diatom, Thalassiosira nordenskioldi, as a food resource. Nauplii newly hatched from eggs reached the adult stage in ca. 150 days (5°C) with a sigmoidal developmental pattern and no sexual difference in development pattern. Starvation experiments indicated that the starved copepodids (C1–C4) became more vulnerable to high temperature with the progression of developmental stage, suggesting that the post-bloom condition with low food availability and increased temperature is harsh for their copepodids. The results of this study in conjunction with previous findings suggest that E. bungii is well adapted to utilize large-sized phytoplankton, such as a bloom-forming diatoms and, therefore, their recruitment processes, including egg production, development and mortality would be strongly affected by the duration and intensity of the spring bloom.