Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier.

The voltage dependence of capacitance was measured by using the setup which was almost the same as that for the study of ion channels. The coefficient which represents the voltage dependence of capacitance itself also changes as a function of the duration of voltage application if hexadecane is contained in bilayer lipid membrane (BLM). The method of Alvarez, O., and R. Latorre (1978. Biophys. J. 21:1-17) was extended to treat BLM with hexadecane.     

Lake restoration by biomanipulation using piscivore and Daphnia stocking; results of the biomanipulation in Japan

Biomanipulation has been employed in numerous locations throughout the world as a means for reducing phytoplankton biomass; however, it has not been employed very often in Japan. A common approach involves the introduction of piscivorous fish to reduce the abundance of planktivorous fish. In our study, to first apply biomanipulation, we stocked Lake Shirakaba (a high-altitude, protected area in a park) in central Japan with rainbow trout fingerlings and cladoceran Daphnia (Daphnia galeata) in 2000. A “pre-biomanipulation” data set (1997–1999) and “a post-biomanipulation” data set (2000–2006) allowed us to evaluate the lake's response to biomanipulation. After the biomanipulation, zoo-planktivorous pond smelt disappeared and a large population of Daphnia had been established, which substantially reduced the number of the previously dominant small cladocerans and rotifers. Water transparency increased from about 2 m (before biomanipulation) to more than 4 m (after biomanipulation). Reductions in algal biomass and increased transparency led to expansion of the submerged macrophyte Elodea nuttallii. Total phosphorus concentrations declined as well over this time period. Based on these results, we concluded that biomanipulation using piscivore and Daphnia stocking succeeded in improving lake water quality by reducing algal abundance and providing favorable conditions for the establishment of rooted plants.     

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.     

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus

ABSTRACT

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2–7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1–7) and the chemically prepared compounds (8–10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3–10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1–10 showed significant inhibition activity against Axl and/or immune checkpoint.     

The fission yeast gamma-tubulin complex is required in G(1) phase and is a component of the spindle assembly checkpoint

Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the gamma-tubulin complex. We have identified fission yeast Alp4 and Alp6, which are homologues of the gamma-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc. Spc98/Hs.Gcp3, respectively. The size of the fission yeast gamma-tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects. First, monopolar mitotic spindles form. Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G(1) phase and ts mutants become lethal before S-phase. alp4 and alp6 mutants are hypersensitive to the microtubule- destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway.     

Micromechanical evaluation of mineralized multilayers

The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.     

Double meaning of courtship song in a moth

Males use courtship signals to inform a conspecific female of their presence and/or quality, or, alternatively, to ‘cheat’ females by imitating the cues of a prey or predator. These signals have the single function of advertising for mating. Here, we show the dual functions of the courtship song in the yellow peach moth, Conogethes punctiferalis, whose males generate a series of short pulses and a subsequent long pulse in a song bout. Repulsive short pulses mimic the echolocation calls of sympatric horseshoe bats and disrupt the approach of male rivals to a female. The attractive long pulse does not mimic bat calls and specifically induces mate acceptance in the female, who raises her wings to facilitate copulation. These results demonstrate that moths can evolve both attractive acoustic signals and repulsive ones from cues that were originally used to identify predators and non-predators, because the bat-like sounds disrupt rivals, and also support a hypothesis of signal evolution via receiver bias in moth acoustic communication that was driven by the initial evolution of hearing to perceive echolocating bat predators.     

Design and synthesis of DPP-IV inhibitors lacking the electrophilic nitrile group

A series of (4beta-substituted)-L-prolylpyrrolidine analogs lacking the electrophilic nitrile function were synthesized and their dipeptidyl peptidase IV (DPP-IV) inhibitory activity and duration of ex vivo activity were evaluated. Structural optimization of a N-(3-phenyl-1,2,4-thiadiazol-5-yl)piperazine analog 8, which was found by high-speed analog synthesis, was carried out to improve the potency and duration of action. A representative compound 26 was evaluated to assess its effect on the plasma glucose level after the oGTT (oral glucose tolerance test) in normal rats. Structure-activity relationships (SAR) are also presented.     

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

1. Download : Download full-size image

     

Merkel cell-nerve cell interaction undergoes formation of a synapse-like structure in a primary culture

Merkel cells have been assumed to guide nerve fibers to the skin. However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable established culture system of Merkel cells. Here we show that Merkel cells isolated from rat footpad skin were successfully cultured in a monolayer with keratinocytes. Keratinocytes did not affect any structural changes in Merkel cells. When nerve cells (NG108-15 or PC12) were added to the culture system, both nerve fibers and cytoplasmic processes of Merkel cells outgrew and cooperatively organized synapse-like structures at their contact points. Nerve cells promoted Merkel cell survival, compared with keratinocytes only. Merkel cell proliferation was not detected in all conditions, even with nerve growth factor, neurotrophin-3, interleukin-6 and tumor necrosis factor-alpha. The data suggest, firstly, that Merkel cells may guide nerve fibers to the skin by interacting with nerve cells; and, secondly, that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells, although Merkel cells may be a terminally differentiated cell type. Our method could open a way to study Merkel cell biology.