Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a co-culture system using retinal capillary endothelial cell line (TR-iBRB2)

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.     

Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.     

Intragastric administration of allyl isothiocyanate increases carbohydrate oxidation via TRPV1 but not TRPA1 in mice

The transient receptor potential (TRP) channel family is composed of a wide variety of cation-permeable channels activated polymodally by various stimuli and is implicated in a variety of cellular functions. Recent investigations have revealed that activation of TRP channels is involved not only in nociception and thermosensation but also in thermoregulation and energy metabolism. We investigated the effect of intragastric administration of TRP channel agonists on changes in energy substrate utilization of mice. Intragastric administration of allyl isothiocyanate (AITC; a typical TRPA1 agonist) markedly increased carbohydrate oxidation but did not affect oxygen consumption. To examine whether TRP channels mediate this increase in carbohydrate oxidation, we used TRPA1 and TRPV1 knockout (KO) mice. Intragastric administration of AITC increased carbohydrate oxidation in TRPA1 KO mice but not in TRPV1 KO mice. Furthermore, AITC dose-dependently increased intracellular calcium ion concentration in cells expressing TRPV1. These findings suggest that AITC might activate TRPV1 and that AITC increased carbohydrate oxidation via TRPV1.     

On the synchorological and floristic trends and discontinuities in regard to the Japan-Liukiu-Formosa area

Conclusion and summary The author discussed the floristic and vegetational differences (a) between Formosa and the Philippines, especially between Botel Tobago and Formosa, (b) between Formosa and Continental China, (c) between Japan proper and Continental China together with Corea, Mandschuria and the eastern part of Siberia, (d) between Formosa and Japan proper, and then he discussed (e) the flora of the Liukius, especially the differences between the Yaeyama Islands and Formosa as well as between Yaku Shima and the Amami Islands. In addition he briefly outlined the vegetation of Formosa and Japan, especially the altitudinal distribution of various forest-communities.The conclusions confirmed the floristic discontinuity between Formosa and the Philippines already set forth by E. D. Merrill and several other botanists. Concerning the phytogeographical position of Botel Tobago, the writer confirms the opinion of T. Kano and R. Kanehira that the flora of Botel Tobago is an extension of that of the Batan-Babuyan Islands, the northern border of the Philippines.The floristic close-relation between Japan proper and Continental China is evident from the phytogeographical distribution of the genera of higher plants found in Japan.Data show that the Formosan flora, especially of the highland, is more closely related to the south-western part of China, and also to the Himalayan region, than to the other surrounding regions.The writer considers that the relationship of the flora of Japan proper to that of Formosa is rather weak. He thinks, however, that their floras and vegetations are related to each other through their mutual relation to the lowland and lower montane flora of Continental China situated besides both of them.It is concluded that the flora of the Liukius is an extension of the Formosan flora, judging by the distribution of the genera and species. Looking over the Japan-Liukiu-Formosa area, a conspicuous floristic and synchorological discontinuity is recognized between Yaku Shima and the Amami Islands, and it is conceivable that the Yaku Shima flora is a south-western extension of the flora of Kyushu or Japan proper, as well as the vegetation of Yaku Shima is closely related essentially to that of Japan proper.The writer and his cooperators adopted a new name of Ardisieto-Shiietum Sieboldi to the Shiia forest of Yaku Shima.The writer is unable to make any synchorological or plant-sociological precise comparison between Japan and Continental China along with the eastern part of Siberia and moreover between Formosa and Continental China, because of few or no plant-sociological detailed data of the continental area in the Far Eastern Asia.The author delivered a special lecture on this subject before the botany class in the National Taiwan University, Taipei, Formosa (Taiwan), on December 4^th 1953. — Contributions from the Department of Biology, Faculty of Science, Kyushu University, No. 38.     

Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation

The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.     

Application of menthol to the skin of whole trunk in mice induces autonomic and behavioral heat-gain responses

When ambient temperature is decreased in mammals, autonomic and behavioral heat-gain responses occur to maintain their core temperatures. However, what molecules in cutaneous sensory nerve endings mediate cooling-induced responses is unclear. Recently, transient receptor potential melastatin-8 (TRPM8) has been identified in cell bodies of sensory neurons as low-temperature and menthol-activated cation channel. We hypothesized that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses. To activate TRPM8 specifically, we applied 1-10% menthol to the skin of whole trunk in mice instead of cooling and measured core temperatures and autonomic and behavioral heat-gain responses. Solvent of menthol (100% ethanol) was used as control. Significant elevation of core temperatures was observed between 20 and 120 min after menthol application. Pretreatment with diclofenac sodium, an antipyretic drug, did not affect this hyperthermia, indicating that the menthol-induced hyperthermia is not fever. Menthol application induced a rise in oxygen consumption, shivering-like muscle activity, tail skin vasoconstriction (autonomic responses), and heat-seeking behavior. All of them are typical heat-gain responses. These results support the hypothesis that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses.     

Detection of single-base mismatch at distal end of DNA duplex by electrochemical impedance spectroscopy

Herein, we report an anomalous electrochemical behavior of surface-bound DNA duplex that has single-base mismatches at its distal end. Single-stranded 15-base DNA was immobilized at its 5'end onto gold electrode surfaces. After hybridization with complementary or mismatched DNA, electrochemical impedance spectra were obtained using [Fe(CN)(6)]3-/4- as redox marker ions. Hybridization with the complementary DNA reduced the charge-transfer resistance (R(CT)), whereas single-base mismatches at the distal end of the duplex largely increased the R(CT). This anomaly was found only with the distal end: the increase in R(CT) was not observed for mismatches at either the middle or the proximal end. These results indicate that electrochemical detection of single-base alterations at an end of sample DNA is exceptionally easy because of the diametrically opposite responses. This detection principle is promising for the typing of single-nucleotide polymorphisms in combination with the single-base primer extension protocol.     

17,20-lyase inhibitors. Part 4: design, synthesis and structure-activity relationships of naphthylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of naphthylmethylimidazole derivatives and related compounds have been investigated as selective 17,20-lyase inhibitors. Optimization of the substituent at the 6-position on the naphthalene ring was performed to yield a methylcarbamoyl derivative, which exhibited potent inhibitory activity against human 17,20-lyase and promising selectivity (>200-fold) for 17,20-lyase over CYP3A4. Further modifications of the methylcarbamoyl derivative led to the discovery of the corresponding tricyclic compound, which showed highly potent activity against human 17,20-lyase (IC(50) 19 nM) and good selectivity (>1000-fold) for inhibition of 17,20-lyase over CYP3A4. Additional biological evaluation revealed that the tricyclic compound had potent in vivo efficacy in monkeys and favorable pharmacokinetic profiles when administered in rats. Asymmetric synthesis of the selective tricyclic inhibitor was also achieved using a chiral α-hydroxy ketone.     

Automatic sleep/wake scoring from body motion in bed: validation of a newly developed sensor placed under a mattress

The purpose of this study was to formulate a "sleep/wake" scoring algorithm for processing activity measurements obtained using a newly developed nonwear actigraphy (NWA) device, and to test its validity. The NWA device has a highly sensitive pressure sensor and is placed under a mattress. It can continuously record the activity of a person lying on the mattress and identify an "in-bed/out-of-bed" state from the vibrations of the mattress. We formulated the sleep/wake scoring algorithm by using data obtained simultaneously by wrist actigraphy (Act) and the NWA device in 33 healthy participants. Agreement rate, sensitivity, and specificity with Act were 95.7%, 97.6%, and 75.8% (33 healthy people); the corresponding values were 85.9%, 89.1%, and 79.8% for 12 nursing home residents and 93.7%, 97.2%, and 60.8% for 60 nights for 6 healthy persons who slept 10 nights on their futons. Agreement rate, sensitivity, and specificity with polysomnography were in almost perfect agreement with Act (12 nights; 6 healthy persons who slept 2 nights). All our validation results indicate that the NWA device, placed under a mattress or a futon, can produce almost identical sleep/wake scores to Act. It is expected that the NWA device, a nonwear device for scoring sleep/wake and in-bed/out-of-bed, enables convenient long-term sleep-related evaluation in various fields, including hospital settings, home-care settings, and care facility settings such as nursing homes.