Hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) and methyl-β-cyclodextrin (MβCD) down regulate caveolin expression in human lens epithelial cells (HLECs)

Oxidative stimulation induced by hydrogen peroxide (H₂O₂) on human lens epithelial cells (HLECs) was performed to observe the effects on cell viability and caveolin expression, and cholesterol depletion in HLECs caused by methyl-β-cyclodextrin (MβCD) was also studied. SRA01/04 HLECs were exposed to H₂O₂ or MβDC of various concentrations and durations. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the effect of H₂O₂ on the viability of SRA01/04 HLECs. The distributions of caveolins after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Immunoblotting was performed to analyze alterations of caveolins expression. We observed that the viability of SRA01/04 HLECs under 0.1 mM H₂O₂ for 10 min or longer was significantly reduced (*p < 0.05, F = 11.63). Laser scanning microscopy showed immunofluorescent caveolins in SRA01/04 HLECs under 0.1 mM H₂O₂ for 10 min or longer; caveolins were largely confined to intracellular domains. Western blots showing both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5, or 1.0 mM H₂O₂ for 30 min were significantly reduced, compared with the untreated (*p < 0.05, F = 6.149, or *p < 0.05, F = 14.489, respectively). In addition, the membrane and total caveolin protein level after being treated with 0.1 mM H₂O₂ (*p < 0.05, F = 6.843, or *p < 0.05, F = 7.944, respectively) for different durations also down regulated. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. This study concludes that the responses of HLECs to oxidative stress may include down regulation of caveolin and phosphorylation of caveolin-1 on tyrosine 14 and that MβCD also down regulates caveolin while depleting cholesterol in HLECs. Published in Russian in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 6, pp. 994–1001. The article was submitted by the authors in English.     

Structure of Deoxyhaemoglobin Zürich (HisE7(63β) — > Arg)

The conformation of Arginine E7 in the mutant Deoxyhaemoglobin Zürich differs from that found in the carbonmonoxy form. Comparison with the structure of normal haemoglobin-A allows the changes in oxygen affinity and binding kinetics to be rationalized.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Evolution of mixed-linkage (1 -> 3, 1 -> 4)-β-D-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes

Background and Aims

Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage (1 → 3, 1 → 4)-β-d-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicelluloses occurring during Equisetum evolution.

Methods

Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endoglucanase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer chromatography.

Key Results

Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharide ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xyloglucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosylated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides.

Conclusions

G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the structure and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit sizes.     

乙酰胆碱酯酶(AChE)与β淀粉样肽(Aβ)的相互关系

老年性痴呆症(Alzheimer‘s Disease,AD)是一种慢性的、进行性的神经系统退行性疾病。主要特征为各方面智力的损伤,包括学习记忆、语言、读写、行为,以及对周围环境的识别,最终可导致死亡。它有三大病理特征：(1)主要由β淀粉样肽(β—amyloid peptide,Aβ)沉积而成的淀粉样斑;(2)由高度磷酸化的Tau蛋白组成的神经纤维缠结;(3)神经元及神经突触的丢失。在AD疾病的发病机制及治疗的对策中,AD和乙酰胆碱酯酶(acetylcholinesterase,AChE)都有重要的作用。近年来对Aβ和AChE在AD疾病发生发展过程中相互作用的研究有了越来越多的报道。在此,作者对Aβ和AChE的关系作一综述。     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

A new synthesis and electrochemistry of 1,1′-bis(β-hydroxyethyl)ferrocene

The preparation of 1,1′-bis(β-hydroxyethyl)ferrocene (1) by oxidation of 1,1′-divinylferrocene is described. Compound 1 has been characterized by ¹H and ¹³C{¹H} NMR, and cyclic voltammetry. The electrochemical data are compared to ferrocene and the closely related 2-ferrocenylethanol, 2.     

Divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms are less preferred than divalent zinc ion bound amyloid-β(40) and amyloid-β(42) alloforms

Divalent copper and zinc ions bind to the amyloid-β(40) and amyloid-β(42) alloforms and affect their structural stability as well as their chemical and physical properties. Current literature debates the impact of copper ions on amyloid-β alloforms. Recently, we reported the structural and thermodynamic properties of apo amyloid-β and divalent zinc ion bound amyloid-β alloforms (see, Wise-Scira et al. in J Biol Inorg Chem 17:927–938, 2012 and Coskuner et al. in ACS Chem Neurosci 4: 310–320, 2013). In our search for understanding the impacts of transition metal ions on disordered amyloid-β, we also developed and reported new potential functions using quantum mechanics, which are required for high-quality molecular dynamics simulations of divalent copper ion bound amyloid-β alloforms (see, Wise and Coskuner in J Comput Chem 35:1278–1289, 2014). The structures and thermodynamic properties of the divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms in an aqueous medium are studied. The secondary and tertiary structures of divalent copper ion bound amyloid-β(40) and amyloid-β(42) along with their thermodynamic properties including enthalpy, entropy, Gibbs free energy and potential of mean force surface are investigated. Results are compared to those for apo amyloid-β and divalent zinc ion bound amyloid-β alloforms. Results demonstrate that copper binding to Aβ alloforms is thermodynamically less preferred rather than zinc binding. Less compact structures of copper ion bound amyloid-β alloforms possess reduced stability in comparison to zinc ion bound amyloid-β alloforms. Cu(II) binding impacts the thermodynamic properties, secondary and tertiary structural properties of Aβ40 and Aβ42.     

Antifouling poly(β-peptoid)s

A new type of polymer highly resistant to nonspecific protein adsorption is reported. Poly(N-methyl-β-alanine) (PMeA) and poly(N-ethyl-β-alanine) (PEtA) synthesized via cobalt-catalyzed carbonylative polymerization of N-methylaziridine and N-ethylaziridine were end-functionalized with thiol groups and grafted onto Au surfaces. Protein adsorption was studied by the surface plasmon resonance (SPR) method. The amounts of representative single proteins adsorbed onto the PMeA- and PEtA-grafted surfaces were below the detection limit of SPR at the pg/mm(2) level. After exposure to full blood plasma and serum for 10 min, protein adsorption was at the level of ～ 100 pg/mm(2), similar to the level of protein adsorption on poly(ethylene glycol) surfaces subjected to identical conditions. These poly(β-peptoid)s therefore provide excellent protein resistance comparable to the best antifouling materials known to date. The strong proton-accepting ability when forming hydrogen bonds is suggested to be an important attribute for these poly(β-peptoid)s as well as other poly(tertiary amide)s as antifouling materials.     

β-Diiminatolanthanoid(III) halides revisited

The crystalline compounds [LnCl₂(L)(thf)₂] [Ln = Ce (1), Tb (2), Yb (3)], [NdI₂(L)(thf)₂] (4), [LnCl(L′)₂] [Ln = Tb (5), Yb (6) (a known compound)] and [YbCl(L′′)(μ-Cl)₂Li(OEt₂)₂] (7) have been prepared [L = {N(C₆H₃Prⁱ₂-2,6)C(H)}₂CPh, L′ = {N(SiMe₃)C(Ph)}₂CH, L′′ = {N(SiMe₃)C(C₆H₄Ph-4)}₂CH]. The X-ray molecular structures of 2-7 have been established; in each, the monoanionic ligand L, L′ or L′′ is N,N′-chelating and essentially π-delocalised. Each of 1-7 was prepared from the appropriate LnCl₃, or for 4 [NdI₃(thf)₂], and an equivalent portion of the appropriate alkali metal [Li for 7, Na for 2, 3 and 5, or K for 1, 4 and 6] β-diiminate in thf; the isolation of exclusively 5 and 6 (rather than the L′ analogues of 2 or 3) is noteworthy, as is the structure of 7 which has no precedent in Group 3 or 4f metal β-diiminato chemistry.     

Poly(β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) (PMLA) from <Emphasis Type="Italic">Aureobasidium</Emphasis> spp. and its current proceedings

Poly(β-l-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(β-l-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.     

β-内酰胺酶(β-Lactamase)抑制剂的抑菌试验

自从抗生素被发现以来,许多传染病都得到了控制。然而随着抗生素的广泛应用,尤其是不合理的滥用,微生物对抗生素的耐药性问题也越趋严重,耐青霉素的细菌已达90％。对广泛使用的β-内酰胺类抗生素(青霉素、头孢霉素)的主要耐     

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

β-β交联的Fe(Ⅱ)-Co(Ⅱ)杂化血红蛋白玻尔(Bohr)效应的研究

本文使用500MHz ~1H NMR谱仪研究了β—β交联的β_1 82Lys—β_2 82Lys不对称Fe(Ⅱ)Co(Ⅱ)杂化血红蛋白在去氧状态下其四级结构与溶液 pH的相互关系.实验结果证明,溶液中H~+浓度的减少,有利于血红蛋白从束缚态(T态)向松弛态(R态)方向转化.在可交换质子区(9.0—15.0PPm)由pH的改变所引起的[2(Co)β(Fe)]_A[α(Co)β(Co)]_cXL的~1H NMR谱峰的变化比[α(Fe)β(Co)]_A[α(Co)β(Co)]_cXL的~1H NMR谱峰的变化大.这为研究血红蛋白四个亚基的构象随pH改变而变化的先后次序提供了一定的依据,可以认为,当溶液中H~+浓度减少时,首先使蛋白构象发生改变的应在α—亚基上,而不在β—亚基上.此外,从它们的~1H NMR谱图随pH的变化可推测,溶液中除了 T态和R态之外,还存在着一些中间过渡态.o     

Valbro : un nouveau site à vertébrés de l’Oligocène inférieur (MP 22) de France (Quercy). II – Oiseaux (Aves)

The bird remains from Valbro belong only to three taxa, Paraortyx lorteti, Archaeotrogon venustus, and A. zitteli. This association makes it possible to ascribe to this locality an early Oligocene age, reference-levels MP 22 or MP 23, which is in agreement with the age given by the mammals.     

Synthetic mucin fragments. Methyl 6-O-(2-acetamido-2-deoxy-β-D-glycopyranosyl)-β-D-galactopyranoside,methyl 3,4-di-O-(2-acetamido-2-deoxy-β-D-glycopyranosyl)-β-D-galactopyranoside,and methyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 å 3)-O-β-D-galactopyranosyl-(1 å 3)-O-(2-acetamido-2-deoxy-β- D-glucopyranosyl)-(1 å 3)-β-D-galactopyranoside

Condensation of methyl 2,6-di-O-benzyl-β-d-galactopyranoside with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)-[2,1,-d]-2-oxazoline (1) in 1,2-dichloroethane, in the presence of p-toluenesulfonic acid, afforded a trisaccharide derivative which, on deacetylation, gave methyl 3,4-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2,6-di-O-benzyl-β-d- glactopyranoside (5). Hydrogenolysis of the benzyl groups of 5 furnished the title trisaccharide (6). A similar condensation of methyl 2,3-di-O-benzyl-β-d-galactopyranoside with 1 produced a partially-protected disacchraide derivative, which, on O-deacetylation followed by hydrogenolysis, gave methyl 6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-glactopyranoside (10). Condensation of methyl 3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-benzyl-β-d- galactopyranoside with 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of powdered mercuric cyanide gave a fully-protected tetrasaccharide derivative, which was O-deacetylated and then subjected to catalytic hydrogenation to furnish methyl O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-O-β-d-galactopyranosyl-(1å3)-O-(2-acetamido-2-deoxy- β-d-glucopyranosyl)-(1å3)-β-d-galactopyranoside (15). The structures of 6, 10, and 15 were established by ¹³C-n.m.r. spectroscopy.     

Synthesis of 1-(2,3-Dideoxy-β-d-glycero-pent-2-enofuranosyl)thymine (d4T; Stavudine) from 5-Methyluridine

Abstract

A practical synthetic method of d4T (3) from 5-methyluridine (2a) was developed. The Marumoto-Mansuri method was modified using 2′,3′-O-methoxy-ethylidene-5-methyluridine (10) as an intermediate to afford 1-(3,5-di-O-acetyl-2-bromo-2-deoxy-β-D-ribofuranosyl)thymine (6a) in high yield with less formation of by-products. The reaction mechanism was also discussed.