Humanin Inhibits Neuronal Cell Death by Interacting with a Cytokine Receptor Complex or Complexes Involving CNTF Receptor α/WSX-1/gp130

Humanin (HN) inhibits neuronal death induced by various Alzheimer''s disease (AD)-related insults via an unknown receptor on cell membranes. Our earlier study indicated that the activation of STAT3 was essential for HN-induced neuroprotection, suggesting that the HN receptor may belong to the cytokine receptor family. In this study, a series of loss-of-function tests indicated that gp130, the common subunit of receptors belonging to the IL-6 receptor family, was essential for HN-induced neuroprotection. Overexpression of ciliary neurotrophic factor receptor α (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it. These results suggest that both CNTFR and WSX-1 may be also involved in HN binding to cells. Consistent with these results, loss-of-functions of CNTFR or WSX-1 in neuronal cells nullified their responsiveness to HN-mediated protection. In vitro–reconstituted binding assays showed that HN, but not the other control peptide, induced the hetero-oligomerization of CNTFR, WSX-1, and gp130. Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130.     

Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.     

<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb. α-mannosidase belongs to the glycosyl hydrolase family 38

α-Mannosidase (EC 3.2.1.24) was purified from ‘Iseimo’, a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting α-mannosidase activity. We purified α-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65°C. The K _m value for p-nitrophenyl-α-d-mannopyranoside was 0.35 ± 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved α-1,2 linkages preferentially to α-1,6 and α-1,3 linkages. The M _r of purified α-mannosidase was estimated to be 250–260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme’s properties indicated that Iseimo α-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.     

Electrophoretic evidence for homoeologous chromosome pairing in the apogamous fern species<Emphasis Type="Italic"> Dryopteris nipponensis</Emphasis> (Dryopteridaceae)

Segregation of genotypes through homoeologous chromosome pairing in the apogamous species Dryopteris nipponensis was tested by electrophoretic analysis. Of 284 progeny examined (250 gametophytes and 34 sporophytes), from the parental sporophyte with the Pgi-2 genotype abc, five showed different genotypes from that of the parent (three aac, one bbc and one bcc). This is the first evidence for genetic segregation in the progeny of apogamous fern species. Electronic Publication     

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.     

ULTRASTRUCTURAL STUDY ON SPORE WALL MORPHOGENESIS IN LYCOPODIUM CLAVATUM (LYCOPODIACEAE)

Spore wall morphogenesis of Lycopodium clavatum was observed by transmission electron microscopy. The spore plasma membrane indicates the reticulate spore sculpture shortly after meiosis. The mature spore wall of this species consists of two layers, inner endospore and outer exospore. There is no perispore in the sporoderm of this species. The exospore formation begins during the tetrad stage; and this layer is divided into two distinct sublayers, an outer lamellar layer and an inner granular layer. The lamellar layer is formed on the sculptured spore plasma membrane. Additional lamellae attach to this layer in a centripetal direction. For that reason, this layer may be derived from spore cytoplasm. The granular layer is formed only in the proximal region following lamellar layer formation, and it also may be derived from spore cytoplasm. The endospore is formed lastly and seems to be derived from spore cytoplasm as well. Accordingly, the spore sculpture of this species may be under the genetic control of the spore nucleus.     

Preparation and biodegradability of chitin derivatives having mercapto groups

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

An early effect of aflatoxin B1 administered in vivo on the growth of bone marrow CFU-GM and the production of some cytokines in rats

Our data demonstrate the granulopoietic toxicity of aflatoxin B₁ (AFB₁)in vivo and show an impact of this mycotoxin on the production of some humoral regulatory factors dealing with the granulopoietic developmental pathway (CSA, IL-1, IL-2). The dose of AFB₁ studied represented approximately 1/5 of LD₅₀ for young male rats. An early suppressive effect of AFB₁ towards CFU-GM was transient in treated animals. The peak in granulopoietic activity was preceded in time by an increased CSA and IL-1 formation. Elevated IL-2 synthesis and increased T cell activation paralleled the peak in granulopoietic activity.Abbreviations AFB₁ Aflatoxin B₁ - CFU-GM granulocyte-monocyte colony-forming unit - CSA colony-stimulating activity - CSF colony-stimulating factor - GM-CSF granulocyte-monocyte CSF - G-CSF granulocyte CSF - M-CSF monocyte CSF - IL-1–6 Interleukin 1–6 - TNF tumour necrosis factor - IFN Interferon     

Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system

Lysoglycosphingolipids were produced from glycosphingolipids by using sphingolipid ceramide N-deacylase, which cleaves the N-acyl linkage between fatty acids and sphingosine bases in various glycosphingolipids. The enzyme reaction was done in a biphasic media prepared with water;-immiscible organic solvent and aqueous buffer solution containing the enzyme. We investigated the effects of organic solvents and detergents on lysoglycosphingolipid production in the biphasic system. Among the organic solvents tested, n-butylbenzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-heptadecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis. Hydrolysis of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addition of n-decane. The hydrolytic activity of the enzyme was enhanced by the addition of 0.8% sodium taurodeoxycholate or sodium cholate to the aqueous phase. The most effective hydrolysis of various glycosphingolipids by the enzyme was thus obtained in the aqueous-n-decane biphasic system containing 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids released from GM1 by the action of the enzyme were trapped and diffused into the organic phase, while lysoGM1 remained in the aqueous phase.Thus the almost complete hydrolysis of GM1 was achieved using the biphasic system, while at most 70% of hydrolysis was obtained using normal aqueous media possibly due to the inhibition of hydrolysis reaction by accumulation of fatty acids in the reaction mixture.