早川 枝李

Although the neuronal protein α-synuclein (α-syn) is thought to play a central role in the pathogenesis of Parkinson's disease (PD), its physiological function remains unknown. It is known that α-syn is also abundantly expressed in erythrocytes. However, its role in erythrocytes is also unknown. In the present study, we investigated the localization of α-syn in human erythroblasts and erythrocytes. Protein expression of α-syn increased during terminal differentiation of erythroblasts (from day 7 to day 13), whereas its mRNA level peaked at day 11. α-syn was detected in the nucleus, and was also seen in the cytoplasm and at the plasma membrane after day 11. In erythroblasts undergoing nucleus extrusion (day 13), α-syn was detected at the periphery of the nucleus. Interestingly, we found that recombinant α-syn binds to trypsinized inside-out vesicles of erythrocytes and phosphatidylserine (PS) liposomes. The dissociation constants for binding to PS/phosphatidylcholine (PC) liposomes of N-terminally acetylated (NAc) α-syn was lower than that of non NAc α-syn. This suggests that N-terminal acetylation plays a significant functional role. The results of the present study collectively suggest that α-syn is involved in the enucleation of erythroblasts and the stabilization of erythroid membranes.

Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.

2011年1月から2014年12月までに医動物学部門へ照会された120症例について報告する。マラリア根治療法前(46例)とマラリア以外の疾患治療前や溶血性疾患除外目的(14例)にglucose-6-phosphate-dehydrogenase(G6PD)欠損症スクリーニング依頼があり、新生児黄疸を示したコートジボワール出身の男児と、フィリピン出身の母親と息子2人に欠損症を認めた。腸管内寄生虫症として日本海裂頭条虫症8例、アジア条虫症2例、アニサキス症6例、蟯虫症2例、鉤虫・糞線虫・回虫症各1例、ヒトブラストシスチス1例が照会され、腸管外寄生虫症としてトキソプラズマ症3例、肺吸虫・肝蛭・マンソン孤虫症各1例が照会された。衛生動物関連疾患9例の内マダニ咬症が6例(発熱1例)を占めた。発熱症例のマラリア除外依頼6例、その他の照会が17例(旅行者下痢症、渡航前相談、寄生虫疾患の除外)であった。当

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI] [Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology. © 2013 Hayakawa et al.

Background: Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes. Methodology and Findings: We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was approximate to 2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO(2) to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes. Conclusion: Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.

Tuberculosis is an infectious and potentially fatal disease caused by the acid-fast bacillus Mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. Lipoarabinomannan ( LAM), an integral component of the MTB cell wall, is released when MTBs are taken into phagosomes and has been reported to be involved in the inhibition of phago-lysosomal (P-L) fusion. However, the physical chemistry of the effects of LAM on lipid membrane structure relative to P-L fusion has not been studied. We produced membranes in vitro composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol to simulate phagosomal lipid membranes and quantified the effects of the addition of LAM to these membranes, using fluorescence resonance energy transfer assays and atomic force microscopy. We found that LAM inhibits vesicle fusion and markedly alters lipid membrane domain morphology and sphingomyelin-chollesterol/dioleoylphosphatidylcholine ratios. These data demonstrate that LAM induces a dramatic reorganization of lipid membranes in vitro and clarifies the role of LAM in the inhibition of P-L fusion and the survival of the MTB within the macrophage.

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6 % complexes of 2-mercapthoethanol (M), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Formation of the inverted hexagonal (H-II) phase from the lamellar (L-alpha) phase of bovine brain-extracted phosphatidylcholine (BBPC) and phosphatidylethanolamine (BBPE) was investigated using P-31-NMR with or without cholesterol. When the ratio of BBPC to BBPE was 1:1, the H-II formation was observed in the presence of 33 mol% cholesterol (i.e., BBPC:BBPE:cholesterol = 1:1:1) at 47 degrees C. The fraction of the H-II phase in the BBPC/BBPE/cholesterol system could be controlled by the addition of dioleoylglycerol. The change of molecular motion of cholesterol affected by the H-II formation was measured at various ratios of the L-alpha to H-II phase with the time-resolved fluorescence depolarization method, using dehydroergosterol as a fluorescent probe. It is observed that the motion of cholesterol became vigorous in the mixture state of the L-alpha, and the H-II phases compared to that in the L-alpha or the H-II phase only. These facts show that cholesterol has the strong ability to induce the H-II phase, probably by special molecular motion, which includes change of its location from the headgroup area to the acyl-chain area.

Cholesterol is highly concentrated in the synaptic membranes which undergo frequent membrane fusion and is believed to play an important role in the lipid phase transition that accompanies fusion. To better understand the phosholipid phase transition and associated molecular motion of phospholipids during the lamellar to inverted hexagonal transition, two major methods were used. First, sn-1,2-dioleoylglycerol was added to synthetic vesicles, and inverted hexagonal formation was detected using P-31-NMR. Multilamellar sheets were composed of 1,2-dioleoyl-3-sn-phosphatidylcholine, 1,2-dioleoyl-3-sn-phosphatidylethanolamine and cholesterol (1:1:1 in molar ratio) and 5 mol%-sn-1,2-dioleoylglycerol was added to transform lamellar to inverted hexagonal structures. However, the transition also required cholesterol. Multilamellar vesicles were composed of phosphatidylcholine, phosphatidylethanolamine and cholesterol and were investigated using a time-resolved fluorescence depolarization method with dansyl-phosphatidylethanolamine as a fluorescent probe. The molecular motion of phospholipid headgroups decreased during the lamellar to inverted hexagonal phase transition, suggesting that the headgroups of phospholipid molecules are more tightly packed in inverted hexagonal structure than in lamellar form of membrane.

To evaluate the diacylglycerol (DG) concentration that may induce the membrane fusion, we examined the formation of the inverted hexagonal (H-II) structure in the multilamellar vesicles comprised of bovine brain phosphatidylcholine (BBPC), bovine brain phosphatidylethanolamine (BBPE) and cholesterol (1:1:1 molar ratio) in the absence or presence of a small amount of dioleoylglycerol (DOG). These lipid layers are considered as the model system for the secreting cell membranes. The formation of the H-II structure was detected using P-31-NMR and fluorescence techniques. The fluorescent probe used was N-(dimethylaminonaphthalene-5-sulfonyl)-dipalmitoylphosphatidylethanolamine (DNS-PE). Without DOG, the H-II structure slightly appeared at 37 degrees C and the extent of the H-II, structure increased with increasing temperature. The onset temperature of the H-II formation decreased to 20 degrees C by the addition of 3 mol % of DOG. Thus, only a few mol % of DG could produce a significant amount of H-II structure, indicating that the physiological amount of DG promotes the fusion of the secreting cell membranes through H-II like intermediates.