Effect of inhaled indomethacin on distilled water-induced airway epithelial cell swelling.

We evaluated the mechanism of the anti-asthmatic effect of inhaled indomethacin (Indo) by using an animal model (guinea pigs) of airway inflammation. After being exposed to either ozone or room air at identical flow rates (5 l/min) for 2 h, guinea pigs were anesthetized, tracheostomized, and lung resistance (RL) was subsequently measured. Guinea pigs inhaled either saline or Indo (1.5 mg/ml) for 1 min before undergoing an ultrasonically nebulized distilled water (UNDW) inhalation test. RL increased significantly after 10 min of UNDW inhalation in the room air and ozone groups but more so in the ozone group. This increase in RL was significantly suppressed by pretreatment with Indo. In the morphometric assessment of airway mucosa, a significant swelling of the epithelial cells after UNDW inhalation was observed in both the room air and ozone groups but especially so in the ozone group. This increase was also suppressed with Indo pretreatment. These results suggest that the increase in RL and the swelling of airway epithelial cells induced by inhaled UNDW in ozone-exposed guinea pigs was suppressed by pretreatment of inhaled Indo and that this suppression may be one of the reasons for the anti-asthmatic effect of inhaled Indo.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.     

A Boolean Function for Neural Induction Reveals a Critical Role of Direct Intercellular Interactions in Patterning the Ectoderm of the Ascidian Embryo

A complex system of multiple signaling molecules often produce differential gene expression patterns in animal embryos. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce Otx expression in the neural lineage at the 32-cell stage. However, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. It is possible that differential activation of one of these signaling pathways is sufficient and the remaining signaling pathways are activated in all cells at similar levels. To address this question, we developed a parameter-free method for determining a Boolean function for Otx expression in the present study. We treated activities of signaling pathways as Boolean values, and we also took all possible patterns of signaling gradients into consideration. We successfully determined a Boolean function that explains Otx expression in the animal hemisphere of wild-type and morphant embryos at the 32-cell stage. This Boolean function was not inconsistent with three sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. These sensing patterns all indicated that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r signaling. To confirm this hypothesis experimentally, we simultaneously knocked-down Admp, Gdf1/3-r, and Fgf9/16/20, and treated this triple morphant with recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity, respectively. Although no differential inputs of Admp, Gdf1/3-r and Fgf9/16/20 signaling were expected under this experimental condition, Otx was expressed specifically in the neural lineage. Thus, direct cell–cell interactions through Efna.d play a critical role in patterning the ectoderm of the early ascidian embryo.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Autotaxin Overexpression Causes Embryonic Lethality and Vascular Defects

Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation     

Occurrence of a novel strain of Scirtothrips dorsalis (Thysanoptera: Thripidae) in Japan and development of its molecular diagnostics

Scirtothrips dorsalis Hood is a cosmopolitan and polyphagous thrips species. Recently, a novel strain of S. dorsalis attacking capsicum crops was found in Japan. A molecular phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I sequences revealed that the capsicum-associated populations were genetically different from Japanese native strains and were closely related to Southeast Asian populations. We named the capsicum-associated populations “strain C” and the Japanese native ones “strain YT”. A total of 10 haplotypes were found in strain C and 26 in strain YT. To differentiate the two strains, we developed a multiplex-PCR method using the ribosomal ITS2 region.     

Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.