Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.     

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice

Aims

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a^−/−) mice and human Akrla-transgenic (Akr1a^tg/+) mice experience different anesthetic actions from pentobarbital—prolonged in Akr1a-knockout (Akr1a^−/−) mice and shortened in human Akrla-transgenic (Akr1a^tg/+) mice.

Main methods

We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice.

Key findings

Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a^−/− mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a^−/− mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.

Significance

Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.     

EVALUATION OF RECOVERABLE FUNCTIONAL LIPID COMPONENTS OF SEVERAL BROWN SEAWEEDS (PHAEOPHYTA) FROM JAPAN WITH SPECIAL REFERENCE TO FUCOXANTHIN AND FUCOSTEROL CONTENTS1

Fucoxanthin (Fx) and fucosterol (Fs) are characteristic lipid components of brown seaweeds that afford several health benefits to humans. This article describes the quantitative evaluation of lipids of 15 species of brown seaweeds with specific reference to Fx, Fs, and functional long‐chain omega‐6/omega‐3 polyunsaturated fatty acids (PUFAs). In addition, fatty‐acid composition of selected species was also accomplished in the study. Major omega‐3 PUFAs in the brown seaweeds analyzed were α‐linolenic acid (18:3n‐3), octadecatetraenoic acid (18:4n‐3), arachidonic acid (20:4n‐6), and eicosapentaenoic acid (20:5n‐3). Both Fx (mg · g^?1 dry weight [dwt]) and Fs (mg · g^?1 dwt) were determined to be relatively abundant in Sargassum horneri (Turner) C. Agardh (Fx, 3.7 ± 1.6; Fs, 13.4 ± 4.4) and Cystoseira hakodatensis (Yendo) Fensholt (Fx, 2.4 ± 0.9; Fs, 8.9 ± 2.0), as compared with other brown seaweed species. Studies related to seasonal variation in Fx, Fs, and total lipids of six brown algae [S. horneri, C. hakodatensis, Sargassum fusiforme (Harv.) Setch., Sargassum thunbergii (Mertens ex Roth) Kuntze, Analipus japonicus (Harv.) M. J. Wynne, and Melanosiphon intestinalis (D. A. Saunders) M. J. Wynne] indicated that these functional lipid components reached maximum during the period between January and March. The functional lipid components present in these seaweeds have the potential for application as nutraceuticals and novel functional ingredients after their recovery.     

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.     

Pyrene-labeled deoxyguanosine as a fluorescence sensor to discriminate single and double stranded DNA structures: Design of ends free molecular beacons

A novel fluorescent DNA probe containing pyrene-labeled C8 alkylamino-substituted 2′-deoxyguanosine was designed in order to discriminate single stranded and double stranded regions in DNA. This fluorescent sensor was used for the design of practically useful 3′- and 5′-ends free self-quenched molecular beacon (MB). Unique MB detectable by pyrene excimer fluorescence was also demonstrated.     

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca²⁺ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H₂O₂ and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k_cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (K_m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.     

Granzyme B-dependent and perforin-independent DNA fragmentation in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes

We previously found that an i.p. injection of anti-CD3 monoclonal antibody (mAb) into mice caused DNA fragmentation in the intestinal villous epithelial cells (IVECs) of the duodenum and the jejunum. In this study, in order to elucidate the mechanism of DNA fragmentation in IVECs, we searched for the inducer(s) of DNA fragmentation by using immunohistochemistry. The release of cytoplasmic granules from intraepithelial lymphocytes (IELs) and the formation of large gaps between IELs and IVECs were observed electron microscopically after antibody administration. The presence and distribution pattern of Granzyme B (GrB), a serine protease in cytolytic granules present in cytotoxic T lymphocytes and natural killer cells and considered to be the responsible molecule for DNA fragmentation in target cells, was examined in detail in intestinal villi by immunohistology. GrB was detected in cytoplasmic granules in nearly all IELs. The time-kinetics of granule release from IELs after mAb injection coincided not only with that of the extracellular diffusion of GrB, but also with that of DNA fragmentation in IVECs. On the other hand, perforin (Pfn), assumed to cooperate with GrB in DNA fragmentation, could not be detected in IELs, and its release was not confirmed after the anti-CD3 mAb injection. Anti-CD3 mAb injection also induced DNA fragmentation in IVECs in Pfn-knockout mice. These results support the notion that DNA fragmentation in IVECs by the stimulated IELs in the present study is induced by a mechanism involving GrB, but independent of Pfn.     

A nocturnal Provespa wasp species as the probable pollinator of epiphytic orchid Coelogyne fimbriata

Many vespid wasps visit flowers to forage nectar. These hymenopterans sometimes contribute to flower pollination. However, none of the nocturnal wasp species is a known pollinator. We collected individuals of light‐attracted Provespa nocturna workers in a montane rainforest on Peninsular Malaysia: some wasps collected bore orchid pollinia on their thoraxes. Among 114 trapped individuals, four bore pollinaria and nine bore only viscidia, suggesting that pollinia had been successfully transported. Molecular barcoding of the pollinia (based on their ITS sequences) assigned the orchid to a species in Coelogyne fimbriata complex. These findings and our other analyses suggest that this nocturnal wasp contributes to pollination of an epiphytic nectarless orchid that probably releases olfactory attractants. This discovery sheds light on the importance of mutualistic relationships between the nocturnal social wasps and epiphytic orchids in Southeast Asian tropical rainforest canopies.