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Summary Hypoxia sensors are essential for regulating local oxygen (O2) homeostasis within the body. This is especially pertinent within the CNS, which is particularly vulnerable to O2 deprivation due to high energetic demand. Here, we reveal hypoxia-monitoring function exerted by astrocytes through an O2-regulated protein trafficking mechanism within the CNS. Strikingly, cultured mouse astrocytes isolated from the parafacial respiratory group (pFRG) and retrotrapezoid nucleus (RTN) region are capable of rapidly responding to moderate hypoxia via the sensor cation channel transient receptor potential (TRP) A1 but, unlike multimodal sensory neurons, are inert to hyperoxia and other TRPA1 activators (carbon dioxide, electrophiles, and oxidants) in normoxia. Mechanistically, O2 suppresses TRPA1 channel activity by protein internalization via O2-dependent proline hydroxylation and subsequent ubiquitination by an E3 ubiquitin ligase, NEDD4-1 (neural precursor cell-expressed developmentally down-regulated protein 4). Hypoxia inhibits this process and instantly accumulates TRPA1 proteins at the plasma membrane, inducing TRPA1-mediated Ca2+ influx that triggers ATP release from pFRG/RTN astrocytes, potentiating respiratory center activity. Furthermore, astrocyte-specific Trpa1 disruption in a mouse brainstem-spinal cord preparation impedes the amplitude augmentation of the central autonomic respiratory output during hypoxia. Thus, reversible coupling of the TRPA1 channels with O2-dependent protein translocation allows astrocytes to act as acute hypoxia sensors in the medullary respiratory center.

Seizures are induced when subjects are exposed to severe hypoxia. It is followed by ventilatory fall-off and eventual respiratory arrest, which may underlie the pathophysiology of death in patients with epilepsy and severe respiratory disorders. However, the mechanisms of hypoxia-induced seizures have not been fully understood. Because astrocytes are involved in various neurological disorders, we aimed to investigate whether astrocytes are operational in seizure generation and respiratory arrest in a severe hypoxic condition. We examined the effects of astrocytic activation blockade on responses of EEG and ventilation to severe hypoxia. Adult mice were divided into two groups; in one group (n = 24) only vehicle was injected, and in the other group (n = 24) arundic acid, an inhibitory modulator of astrocytic activation, was administered before initiation of recording. After recording EEG and ventilation by whole body plethysmography in room air, the gas in the recording chamber was switched to 5% oxygen (nitrogen balanced) until a seizure and ventilatory depression occurred, followed by prompt switch back to room air. Severe hypoxia initially increased ventilation, followed by a seizure and ventilatory suppression in all mice examined. Fourteen mice without arundic acid showed respiratory arrest during loading of hypoxia. However, 22 mice pretreated with arundic acid did not suffer from respiratory arrest. Time from the onset of hypoxia to the occurrence of seizures was significantly longer in the group with arundic acid than that in the group without arundic acid. We suggest that blockade of astrocytic activation delays the occurrence of seizures and prevents respiratory arrest.

The fruit thinning effect of Jasmonic acid derivative, n-propyl dihydrojasmonate (PDJ) on Japanese pear ‘Hosui’ was investigated. During the 1996 season, PDJ at 500, 1000, or 2000 ppm was applied to flower clusters at 17 or 12 days before full bloom (DBFB), at full bloom (FB), or at 7 days after full bloom (DAFB). During the 1998 season, PDJ at 500 or 750 ppm was applied to the whole tree at 18 or 14 DBFB. All PDJ treatments before full bloom induced fruit abscission and resulted in a thinning effect. The best results were obtained with 500 to 750 ppm PDJ applied at 17 or 18 DBFB. In these treatments, the proportion of clusters that had two or fewer fruits was about 64%, compared to the control which was 16%. In addition, the position of dropped fruit on the inflorescence axis in a cluster differed among treatments. In PDJ treatment, from the 1st to 3rd fruits from the base of the floral axis in the cluster tended to abscise easily. Abscission of the 1st and 2nd basal fruits was observed in about 90% of the clusters. There were no significant differences in fruit weight, flesh firmness, brix, and titratable acidity at harvest. PDJ did not inhibit pollen tube growth in the style of the pistil, but in flowers treated with PDJ there were many abnormal ovules at FB. These findings indicate that an application of PDJ at 500 or 750 ppm, 17–18 DBFB budbreak has a consistently good thinning effect. Histological examinations revealed that PDJ causes fruit thinning by preventing fertilization and/or causing seed abortion.

The aim of this study was to compare the results of immunohistochemistry (IHC) assays evaluated by human examiners with the results evaluated by computerized image analysis, and to compare the computerized image analysis results among three automated IHC assays, namely the BioGenex, Dako and Ventana assays. All slides were semiquantitatively evaluated according to the Allred score and J-score by human examiners. The images were analyzed using MacSCOPE version 2.6 for Macintosh according to the H-score and the percentage of positive-stained nuclei per area of carcinoma cells (PP) irrespective of the intensity of the stained nuclei. The H-score for the estrogen receptor (ER) was significantly correlated with the Allred score (P